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Tomoyoshi Nozaki - One of the best experts on this subject based on the ideXlab platform.
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Import of Entamoeba histolytica Mitosomal ATP Sulfurylase Relies on Internal Targeting Sequences
Microorganisms, 2020Co-Authors: Herbert J. Santos, Takashi Makiuchi, Kenichiro Imai, Yoko Chiba, Saki Arakawa, Yoshitaka Murakami, Kentaro Tomii, Tomoyoshi NozakiAbstract:Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that are present in a few species of anaerobic eukaryotes. Similar to other MROs, the Mitosomes of the human intestinal parasite Entamoeba histolytica are highly degenerate, because a majority of the components involved in various processes occurring in the canonical mitochondria are either missing or modified. As of yet, sulfate activation continues to be the only identified role of the relic mitochondria of Entamoeba. Mitosomes influence the parasitic nature of E. histolytica, as the downstream cytosolic products of sulfate activation have been reported to be essential in proliferation and encystation. Here, we investigated the position of the targeting sequence of one of the mitosomal matrix enzymes involved in the sulfate activation pathway, ATP sulfurylase (AS). We confirmed by immunofluorescence assay and subcellular fractionation that hemagluttinin (HA)-tagged EhAS was targeted to Mitosomes. However, its ortholog in the δ-proteobacterium Desulfovibrio vulgaris, expressed as DvAS-HA in amoebic trophozoites, indicated cytosolic localization, suggesting a lack of recognizable Mitosome targeting sequence in this protein. By expressing chimeric proteins containing swapped sequences between EhAS and DvAS in amoebic cells, we identified the ITSs responsible for Mitosome targeting of EhAS. This observation is similar to other parasitic protozoans that harbor MROs, suggesting a convergent feature among various MROs in favoring ITS for the recognition and translocation of targeted proteins.
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an entamoeba specific mitosomal membrane protein with potential association to the golgi apparatus
Genes, 2019Co-Authors: Herbert J. Santos, Kenichiro Imai, Yuki Hanadate, Tomoyoshi NozakiAbstract:The aerobic mitochondrion had undergone evolutionary diversification, most notable among lineages of anaerobic protists. Entamoeba is one of the genera of parasitic protozoans that lack canonical mitochondria, and instead possess mitochondrion-related organelles (MROs), specifically Mitosomes. Entamoeba Mitosomes exhibit functional reduction and divergence, most exemplified by the organelle’s inability to produce ATP and synthesize iron-sulfur cluster. Instead, this organelle is capable of sulfate activation, which has been linked to amoebic stage conversion. In order to understand other unique features and components of this MRO, we utilized an in silico prediction tool to screen transmembrane domain containing proteins in the Mitosome proteome. Here, we characterize a novel lineage-specific mitosomal membrane protein, named Entamoeba transmembrane mitosomal protein of 30 kDa (ETMP30; EHI_172170), predicted to contain five transmembrane domains. Immunofluorescence analysis demonstrated colocalization of hemagglutinin (HA)-tagged ETMP30 with the mitosomal marker, adenosine-5’-phosphosulfate kinase. Mitosomal membrane localization was indicated by immunoelectron microscopy analysis, which was supported by carbonate fractionation assay. Transcriptional gene silencing successfully repressed RNA expression by 60%, and led to a defect in growth and partial elongation of Mitosomes. Immunoprecipitation of ETMP30 from ETMP30-HA-expressing transformant using anti-HA antibody pulled down one interacting protein of 126 kDa. Protein sequencing by mass spectrometry revealed this protein as a cation-transporting P-type ATPase, previously reported to localize to vacuolar compartments/Golgi-like structures, hinting at a possible Mitosome-vacuole/Golgi contact site.
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hetero oligomer of dynamin related proteins participates in the fission of highly divergent mitochondria from entamoeba histolytica
Scientific Reports, 2017Co-Authors: Takashi Makiuchi, Tomoyoshi Nozaki, Herbert J. Santos, Hiroshi TachibanaAbstract:Entamoeba histolytica is an anaerobic parasitic protist and possesses Mitosomes, one of the most highly divergent mitochondrion-related organelles (MROs). Although unique metabolism and protein/metabolite transport machinery have been demonstrated in Entamoeba Mitosomes, the mechanism of mitosomal fusion and fission remains to be elucidated. In this study, we demonstrate that two dynamin-related proteins (DRPs) are cooperatively involved in the fission of Entamoeba Mitosomes. Expression of a dominant negative form of EhDrpA and EhDrpB, and alternatively, repression of gene expression of EhDrpA and EhDrpB genes, caused elongation of Mitosomes, reflecting inhibition of mitosomal fission. Moreover, EhDrpA and EhDrpB formed an unprecedented hetero-oligomeric complex with an approximate 1:2 to 1:3 ratio, suggesting that the observed elongation of Mitosomes is likely caused by the disruption and instability of the complex caused by an imbalance in the two DRPs. Altogether, this is the first report of a hetero-oligomeric DRP complex which participates in the fission of mitochondria and MROs.
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Behavior of DNA-lacking mitochondria in Entamoeba histolytica revealed by organelle transplant
Scientific Reports, 2017Co-Authors: Makoto Kazama, Kumiko Nakada-Tsukui, Tomoyoshi Nozaki, Takashi Makiuchi, Sanae Ogiwara, Kazuhiro Yoshida, Hiroshi TachibanaAbstract:The anaerobic protozoan parasite Entamoeba histolytica has Mitosomes that are mitochondria lacking some canonical functions and organelle DNA. Mitosomes play an important role in the life cycle of the parasite. The distribution of proteins in Mitosomes is not uniform, and how Mitosomes are maintained and retained is unknown. To answer these questions, we developed a transplant method for Mitosomes with hemagglutinin-tagged protein into recipient cells containing Mitosomes with Myc-tagged protein. Immunofluorescence staining showed that the two protein tags colocalized in single Mitosomes in some recipient cells. These results suggest that our transplant method can be used in anaerobic protozoa and that donor Mitosomes may obtain recipient proteins through fusion with other Mitosomes or through de novo synthesis of proteins in recipient cells.
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Screening and discovery of lineage-specific mitosomal membrane proteins in Entamoeba histolytica.
Molecular and Biochemical Parasitology, 2016Co-Authors: Herbert J. Santos, Fumika Mi-ichi, Kenichiro Imai, Yuki Hanadate, Yoshinori Fukasawa, Toshiyuki Oda, Tomoyoshi NozakiAbstract:Entamoeba histolytica, an anaerobic intestinal parasite causing dysentery and extra-intestinal abscesses in humans, possesses highly reduced and divergent mitochondrion-related organelles (MROs) called Mitosomes. This organelle lacks many features associated with canonical aerobic mitochondria and even other MROs such as hydrogenosomes. The Entamoeba Mitosome has been found to have a compartmentalized sulfate activation pathway, which was recently implicated to have a role in amebic stage conversion. It also features a unique shuttle system via Tom60, which delivers proteins from the cytosol to the Mitosome. In addition, only Entamoeba Mitosomes possess a novel subclass of β-barrel outer membrane protein called MBOMP30. With the discoveries of such unique features of Mitosomes of Entamoeba, there still remain a number of significant unanswered issues pertaining to this organelle. Particularly, the present understanding of the inner mitosomal membrane of Entamoeba is extremely limited. So far, only a few homologs for transporters of various substrates have been confirmed, while the components of the protein translocation complexes appear to be absent or are yet to be discovered. Employing a similar strategy as in our previous work, we collaborated to screen and discover mitosomal membrane proteins. Using a specialized prediction pipeline, we searched for proteins possessing α-helical transmembrane domains, which are unique to E. histolytica Mitosomes. From the prediction algorithm, 25 proteins emerged as candidates, two of which were initially observed to be localized to the Mitosomes. Further screening and analysis of the predicted proteins may provide clues to answer key questions on mitosomal evolution, biogenesis, dynamics, and biochemical processes.
Pavel Doležal - One of the best experts on this subject based on the ideXlab platform.
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A Single Tim Translocase in the Mitosomes of Giardia intestinalis Illustrates Convergence of Protein Import Machines in Anaerobic Eukaryotes.
Genome Biology and Evolution, 2018Co-Authors: Eva Pyrihová, Lubos Voleman, Martin Kolisko, Alžběta Motyčková, Natalia Wandyszewska, Radovan Fišer, Gabriela Seydlová, Andrew J. Roger, Pavel DoležalAbstract:Mitochondria have evolved diverse forms across eukaryotic diversity in adaptation to anoxia. Mitosomes are the simplest and the least well-studied type of anaerobic mitochondria. Transport of proteins via TIM complexes, composed of three proteins of the Tim17 protein family (Tim17/22/23), is one of the key unifying aspects of mitochondria and mitochondria-derived organelles. However, multiple experimental and bioinformatic attempts have so far failed to identify the nature of TIM in Mitosomes of the anaerobic metamonad protist, Giardia intestinalis, one of the few experimental models for Mitosome biology. Here, we present the identification of a single G. intestinalis Tim17 protein (GiTim17), made possible only by the implementation of a metamonad-specific hidden Markov model. While very divergent in primary sequence and in predicted membrane topology, experimental data suggest that GiTim17 is an inner membrane mitosomal protein, forming a disulphide-linked dimer. We suggest that the peculiar GiTim17 sequence reflects adaptation to the unusual, detergent resistant, inner mitosomal membrane. Specific pull-down experiments indicate interaction of GiTim17 with mitosomal Tim44, the tethering component of the import motor complex. Analysis of TIM complexes across eukaryote diversity suggests that a "single Tim" translocase is a convergent adaptation of Mitosomes in anaerobic protists, with Tim22 and Tim17 (but not Tim23), providing the protein backbone.
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Giardia intestinalis Mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum
BMC Biology, 2017Co-Authors: Lubos Voleman, Elin Einarsson, Staffan G. Svärd, Jan Tachezy, Vladimíra Najdrová, Ásgeir Ástvaldsson, Pavla Tůmová, Zdeněk Švindrych, Guy M. Hagen, Pavel DoležalAbstract:Background Mitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the Mitosomes, the simplest forms of mitochondria, in the anaerobic protist parasite Giardia intestinalis , a member of the Excavata supergroup of eukaryotes. The Mitosomes have abandoned typical mitochondrial traits such as the mitochondrial genome and aerobic respiration and their single role known to date is the formation of iron–sulfur clusters. Results In live experiments, no fusion events were observed between the Mitosomes in G. intestinalis. Moreover, the organelles were highly prone to becoming heterogeneous. This suggests that fusion is either much less frequent or even absent in Mitosome dynamics. Unlike in mitochondria, division of the Mitosomes was absolutely synchronized and limited to mitosis. The association of the nuclear and the mitosomal division persisted during the encystation of the parasite. During the segregation of the divided Mitosomes, the subset of the organelles between two G. intestinalis nuclei had a prominent role. Surprisingly, the sole dynamin-related protein of the parasite seemed not to be involved in mitosomal division. However, throughout the cell cycle, Mitosomes associated with the endoplasmic reticulum (ER), although none of the known ER-tethering complexes was present. Instead, the ER–Mitosome interface was occupied by the lipid metabolism enzyme long-chain acyl-CoA synthetase 4. Conclusions This study provides the first report on the dynamics of Mitosomes. We show that together with the loss of metabolic complexity of mitochondria, Mitosomes of G. intestinalis have uniquely streamlined their dynamics by harmonizing their division with mitosis. We propose that this might be a strategy of G. intestinalis to maintain a stable number of organelles during cell propagation. The lack of mitosomal fusion may also be related to the secondary reduction of the organelles. However, as there are currently no reports on mitochondrial fusion in the whole Excavata supergroup, it is possible that the absence of mitochondrial fusion is an ancestral trait common to all excavates.
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Giardia intestinalis Mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum.
BMC Biology, 2017Co-Authors: Lubos Voleman, Elin Einarsson, Staffan G. Svärd, Jan Tachezy, Vladimíra Najdrová, Ásgeir Ástvaldsson, Pavla Tůmová, Zdeněk Švindrych, Guy M. Hagen, Pavel DoležalAbstract:Background: Mitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the Mitosomes, the simplest forms of mitochondria, in ...
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Additional file 5: of Giardia intestinalis Mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum
2017Co-Authors: Lubos Voleman, Elin Einarsson, Staffan G. Svärd, Jan Tachezy, Vladimíra Najdrová, Ásgeir Ástvaldsson, Pavla Tůmová, Zdeněk Švindrych, Guy Hagen, Pavel DoležalAbstract:Peripheral Mitosomes divide during all stages of mitosis. (A) G. intestinalis culture was enriched for mitotic trophozoites by albendazole treatment (100 ng/mL) for 6 h at 37 °C. The cells were washed twice in warm medium and fixed, and the Mitosomes were immunolabeled with an anti-GL50803_9296 antibody (red) and stained for nuclei with DAPI (blue). The image represents a deconvolved maximal projection of the Z-stack. Corresponding DIC images are shown. Scale bar, 2 μm. Arrowheads point at dividing Mitosomes. (B) The number of Mitosomes in particular stages of mitosis was determined using fixed cells. The data show a gradual increase in Mitosome number during mitosis. Thirty cells of each mitotic stage were used for the statistics. The error bars represent the standard deviations. (EPS 4730 kb
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Minimal cytosolic iron-sulfur cluster assembly machinery of Giardia intestinalis is partially associated with Mitosomes.
Molecular Microbiology, 2016Co-Authors: Jan Pyrih, Pavel Doležal, Martin Kolisko, Eva Pyrihová, Darja Stojanovová, Somsuvro Basu, Karel Harant, Alexander C. Haindrich, Julius Lukeš, Andrew J. RogerAbstract:Iron-sulfur (Fe-S) clusters are essential cofactors that enable proteins to transport electrons, sense signals, or catalyze chemical reactions. The maturation of dozens of Fe-S proteins in various compartments of every eukaryotic cell is driven by several assembly pathways. The ubiquitous cytosolic Fe-S cluster assembly (CIA) pathway, typically composed of eight highly conserved proteins, depends on mitochondrial Fe-S cluster assembly (ISC) machinery. Giardia intestinalis contains one of the smallest eukaryotic genomes and the Mitosome, an extremely reduced mitochondrion. Because the only pathway known to be retained within this organelle is the synthesis of Fe-S clusters mediated by ISC machinery, a likely function of the Mitosome is to cooperate with the CIA pathway. We investigated the cellular localization of CIA components in G. intestinalis and the origin and distribution of CIA-related components and Tah18-like proteins in other Metamonada. We show that orthologs of Tah18 and Dre2 are missing in these eukaryotes. In Giardia, all CIA components are exclusively cytosolic, with the important exception of Cia2 and two Nbp35 paralogs, which are present in the Mitosomes. We propose that the dual localization of Cia2 and Nbp35 proteins in Giardia might represent a novel connection between the ISC and the CIA pathways.
Alina V. Goldberg - One of the best experts on this subject based on the ideXlab platform.
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Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis
Nature Communications, 2017Co-Authors: Sven-a. Freibert, Alina V. Goldberg, Sabine Molik, Christian Hacker, Paul Dean, Sirintra Nakjang, Shaojun Long, Kacper Sendra, Thomas Williams, Eckhard BillAbstract:Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (Mitosomes) of Trachipleistophora hominis are the functional site of iron-sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that Mitosomes contain a complete pathway for [2Fe-2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron-sulfur protein assembly (CIA) pathway includes the essential Cfd1-Nbp35 scaffold complex that assembles a [4Fe-4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides compelling evidence for the ancient chimeric ancestry of eukaryotes.
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Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis
Nature Communications, 2017Co-Authors: Sven-a. Freibert, Alina V. Goldberg, Sabine Molik, Christian Hacker, Paul Dean, Tom A. Williams, Sirintra Nakjang, Shaojun Long, Kacper Sendra, Eckhard BillAbstract:Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (Mitosomes) of Trachipleistophora hominis are the functional site of iron–sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that Mitosomes contain a complete pathway for [2Fe–2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron–sulfur protein assembly (CIA) pathway includes the essential Cfd1–Nbp35 scaffold complex that assembles a [4Fe–4S] cluster as shown by spectroscopic methods in vitro . Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides compelling evidence for the ancient chimeric ancestry of eukaryotes. The functions of the highly reduced mitochondria (Mitosomes) of microsporidians are not well-characterized. Here, the authors show that the Trachipleistophora hominis Mitosome is the site of iron–sulfur cluster assembly and that its retention is likely linked to its role in cytosolic and nuclear iron–sulfur protein maturation.
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Plasma Membrane-Located Purine Nucleotide Transport Proteins Are Key Components for Host Exploitation by Microsporidian Intracellular
2015Co-Authors: Eva Heinz, Alina V. Goldberg, Robert P. Hirt, Christian Hacker, Paul Dean, Tom A. Williams, Sirintra Nakjang, John Mifsud, Alison Gregory, John M. LucocqAbstract:Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (Mitosomes), consistent with lineage-specific differences in transporter and Mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expresse
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Plasma membrane-located purine nucleotide transport proteins are key components for host exploitation by microsporidian intracellular parasites.
PLOS Pathogens, 2014Co-Authors: Eva Heinz, Alina V. Goldberg, Robert P. Hirt, Christian Hacker, Paul Dean, Sirintra Nakjang, John Mifsud, Alison Gregory, Thomas Williams, John M. LucocqAbstract:Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (Mitosomes), consistent with lineage-specific differences in transporter and Mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.
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Schematic overview of the transporter repertoire of T. hominis.
2012Co-Authors: Eva Heinz, Alina V. Goldberg, Tom A. Williams, Sirintra Nakjang, Christophe J. Noël, Daniel C. Swan, Simon R. Harris, Thomas Weinmaier, Stephanie Markert, Dörte BecherAbstract:An overview of predicted T. hominis transporters with their possible locations in (1) the plasma membrane, (2) the Mitosome, and (3) other endomembranes. The number of predicted proteins of each type is indicated in the icons; the predicted transport substrate (s) are also shown. Details of the predicted enzymes (EC numbers and descriptions) as well as the transporters (TC numbers and descriptions) are provided in the Tables S15 and S8, respectively.
Jan Tachezy - One of the best experts on this subject based on the ideXlab platform.
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Giardia intestinalis Mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum
BMC Biology, 2017Co-Authors: Lubos Voleman, Elin Einarsson, Staffan G. Svärd, Jan Tachezy, Vladimíra Najdrová, Ásgeir Ástvaldsson, Pavla Tůmová, Zdeněk Švindrych, Guy M. Hagen, Pavel DoležalAbstract:Background Mitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the Mitosomes, the simplest forms of mitochondria, in the anaerobic protist parasite Giardia intestinalis , a member of the Excavata supergroup of eukaryotes. The Mitosomes have abandoned typical mitochondrial traits such as the mitochondrial genome and aerobic respiration and their single role known to date is the formation of iron–sulfur clusters. Results In live experiments, no fusion events were observed between the Mitosomes in G. intestinalis. Moreover, the organelles were highly prone to becoming heterogeneous. This suggests that fusion is either much less frequent or even absent in Mitosome dynamics. Unlike in mitochondria, division of the Mitosomes was absolutely synchronized and limited to mitosis. The association of the nuclear and the mitosomal division persisted during the encystation of the parasite. During the segregation of the divided Mitosomes, the subset of the organelles between two G. intestinalis nuclei had a prominent role. Surprisingly, the sole dynamin-related protein of the parasite seemed not to be involved in mitosomal division. However, throughout the cell cycle, Mitosomes associated with the endoplasmic reticulum (ER), although none of the known ER-tethering complexes was present. Instead, the ER–Mitosome interface was occupied by the lipid metabolism enzyme long-chain acyl-CoA synthetase 4. Conclusions This study provides the first report on the dynamics of Mitosomes. We show that together with the loss of metabolic complexity of mitochondria, Mitosomes of G. intestinalis have uniquely streamlined their dynamics by harmonizing their division with mitosis. We propose that this might be a strategy of G. intestinalis to maintain a stable number of organelles during cell propagation. The lack of mitosomal fusion may also be related to the secondary reduction of the organelles. However, as there are currently no reports on mitochondrial fusion in the whole Excavata supergroup, it is possible that the absence of mitochondrial fusion is an ancestral trait common to all excavates.
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Giardia intestinalis Mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum.
BMC Biology, 2017Co-Authors: Lubos Voleman, Elin Einarsson, Staffan G. Svärd, Jan Tachezy, Vladimíra Najdrová, Ásgeir Ástvaldsson, Pavla Tůmová, Zdeněk Švindrych, Guy M. Hagen, Pavel DoležalAbstract:Background: Mitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the Mitosomes, the simplest forms of mitochondria, in ...
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Additional file 5: of Giardia intestinalis Mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum
2017Co-Authors: Lubos Voleman, Elin Einarsson, Staffan G. Svärd, Jan Tachezy, Vladimíra Najdrová, Ásgeir Ástvaldsson, Pavla Tůmová, Zdeněk Švindrych, Guy Hagen, Pavel DoležalAbstract:Peripheral Mitosomes divide during all stages of mitosis. (A) G. intestinalis culture was enriched for mitotic trophozoites by albendazole treatment (100 ng/mL) for 6 h at 37 °C. The cells were washed twice in warm medium and fixed, and the Mitosomes were immunolabeled with an anti-GL50803_9296 antibody (red) and stained for nuclei with DAPI (blue). The image represents a deconvolved maximal projection of the Z-stack. Corresponding DIC images are shown. Scale bar, 2 μm. Arrowheads point at dividing Mitosomes. (B) The number of Mitosomes in particular stages of mitosis was determined using fixed cells. The data show a gradual increase in Mitosome number during mitosis. Thirty cells of each mitotic stage were used for the statistics. The error bars represent the standard deviations. (EPS 4730 kb
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probing the biology of giardia intestinalis Mitosomes using in vivo enzymatic tagging
Molecular and Cellular Biology, 2015Co-Authors: Eva Martincova, Lubos Voleman, Jan Pyrih, Vojtěch žarský, Pavlina Vondrackova, Jan Tachezy, Martin Kolisko, Pavel DoležalAbstract:Giardia intestinalis parasites contain Mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of Mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. An in vivo enzymatic tagging technique based on the Escherichia coli biotin ligase (BirA) has been introduced to G. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import were in vivo tagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific to G. intestinalis and even absent from the related diplomonad parasite Spironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. The in vivo enzymatic tagging also showed that proteins enter the Mitosome posttranslationally in an unfolded state and without vesicular transport.
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The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis
2011Co-Authors: Petr L. Jedelský, Jan Pyrih, Petr Rada, Andrew J. Perry, Neritza Campo Beltrán, Trevor Lithgow, Jan TachezyAbstract: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 Fe
T. Martin Embley - One of the best experts on this subject based on the ideXlab platform.
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Diversity and reductive evolution of mitochondria among microbial eukaryotes
Philosophical Transactions of the Royal Society B, 2010Co-Authors: Karin Hjort, Alina V. Goldberg, Anastasios D. Tsaousis, Robert P. Hirt, T. Martin EmbleyAbstract:All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or Mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and Mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.
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Reductive evolution of the mitochondrial processing peptidases of the unicellular parasites trichomonas vaginalis and giardia intestinalis.
PLOS Pathogens, 2008Co-Authors: Ondřej Smid, Ivan Hrdý, Robert P. Hirt, Simon R. Harris, Anna Matuskova, Tomas Kucera, Marian Novotný, Lenka Horvathova, Eva Kutějová, T. Martin EmbleyAbstract:Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the Mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of Mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia Mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.
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A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi
Nature, 2008Co-Authors: Anastasios D. Tsaousis, Alina V. Goldberg, Robert P. Hirt, Edmund R. S. Kunji, John M. Lucocq, T. Martin EmbleyAbstract:The parasite Encephalitozoon cuniculi contains Mitosomes instead of functional mitochondria. Although mitochondrial carrier proteins are known to be responsible for ATP transport from the mitochondria to the cytosol, Hirt et al . now show this process to be reversed in E. cuniculi , where ATP appears to be transported from the cytosol to the organelle. Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont^ 1 . Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins^ 2 . This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a Mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities^ 2 , 3 , 4 . The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia , to import ATP from the cytosol of their eukaryotic host cells^ 5 , 6 , 7 . The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi Mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi , by reductive evolution and analogous gene replacement. Instead of the Mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle. The micosporidia, including the parasite Encephalitozoon cuniculi that is an opportunistic pathogen in humans, have undergone extreme genomic and cellular reduction. Instead of mitochondria, they contain remnant organelles known as Mitosomes. Somehow, these organisms need to get hold of ATP, and now there is evidence that E. cuniculi uses bacterial-like transport proteins to 'steal' ATP from the cytosol of its eukaryotic host.
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A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi
Nature, 2008Co-Authors: Anastasios D. Tsaousis, Alina V. Goldberg, Robert P. Hirt, Edmund R. S. Kunji, John M. Lucocq, T. Martin EmbleyAbstract:Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont. Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins. This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a Mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities. The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia, to import ATP from the cytosol of their eukaryotic host cells. The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi Mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi, by reductive evolution and analogous gene replacement. Instead of the Mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle.
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Localization and functionality of microsporidian iron–sulphur cluster assembly proteins
Nature, 2008Co-Authors: Alina V. Goldberg, Anastasios D. Tsaousis, Sabine Molik, Karina Neumann, Grit Kuhnke, Frédéric Delbac, Christian P. Vivarès, Robert P. Hirt, Roland Lill, T. Martin EmbleyAbstract:Microsporidia contain a tiny mitochondrial remnant called a Mitosome, but the function of this organelle is unknown. Microsporidian genomes encode several components of the mitochondrial iron–sulphur-cluster machinery. This paper describes the cloning, characterization and subcellular localization of several Fe–S-cluster components for two microsporidia. Although some components localize to the Mitosome, others are cytosolic, raising questions about how their function is coordinated. Microsporidia are highly specialized obligate intracellular parasites of other eukaryotes, including humans. They were long regarded as relics of an early stage of eukaryote evolution, before the acquisition of mitochondria. But recent research has complicated the picture, with the discovery that they contain a tiny mitochondrial remnant, called a Mitosome. The function of the Mitosome is a matter of debate, but microsporidian genomes are known to encode several components of the mitochondrial iron–sulphur-cluster machinery. Golberg et al . now describe the cloning, characterization and subcellular localization of several Fe–S-cluster components for two microsporidia. Although some components localize to the Mitosome, others are cytosolic, raising questions about how their function is coordinated. The data support the suggestion that a key role of the Mitosome is in the biosynthesis of cytosolic Fe–S proteins, including Nar1 and Rli1, that are vital for cell survival. Microsporidia are highly specialized obligate intracellular parasites of other eukaryotes (including humans^ 1 ) that show extreme reduction at the molecular, cellular and biochemical level^ 2 , 3 . Although microsporidia have long been considered as early branching eukaryotes that lack mitochondria^ 4 , they have recently been shown to contain a tiny mitochondrial remnant called a Mitosome^ 2 , 5 . The function of the Mitosome is unknown, because microsporidians lack the genes for canonical mitochondrial functions, such as aerobic respiration and haem biosynthesis. However, microsporidial genomes encode several components of the mitochondrial iron–sulphur (Fe–S) cluster assembly machinery. Here we provide experimental insights into the metabolic function and localization of these proteins. We cloned, functionally characterized and localized homologues of several central mitochondrial Fe–S cluster assembly components for the microsporidians Encephalitozoon cuniculi and Trachipleistophora hominis. Several microsporidial proteins can functionally replace their yeast counterparts in Fe–S protein biogenesis. In E. cuniculi , the iron (frataxin) and sulphur (cysteine desulphurase, Nfs1) donors and the scaffold protein (Isu1) co-localize with mitochondrial Hsp70 to the Mitosome, consistent with it being the functional site for Fe–S cluster biosynthesis. In T. hominis , mitochondrial Hsp70 and the essential sulphur donor (Nfs1) are still in the Mitosome, but surprisingly the main pools of Isu1 and frataxin are cytosolic, creating a conundrum of how these key components of Fe–S cluster biosynthesis coordinate their function. Together, our studies identify the essential biosynthetic process of Fe–S protein assembly as a key function of microsporidian Mitosomes.