Conventional transmission electron microscopy was used to localise double-membrane vesicles probably representing mitochondrial remnants ("mitosomes") in four species of microsporidia. Very few such vesicles were found dispersed throughout cytoplasm with no relationship to other cell organelles. Several double-membrane vesicles per ultrathin section, however, occurred regularly close to the nuclear spindle plaque. These vesicles are identical with the "polar vesicles" typically associated with the microsporidian spindle plaque and known since 1971. The reason for mitosome accumulation near the spindle plaque is unknown. Possibly the spindle plaques are involved in mitosome segregation during cell division.

• MeSH
• aparát dělícího vřeténka metabolismus   ultrastruktura   MeSH
• cytoplazmatické vezikuly metabolismus   ultrastruktura   MeSH
• Microsporidia ultrastruktura   MeSH
• mitochondrie ultrastruktura   MeSH
• transmisní elektronová mikroskopie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes.

• MeSH
• anaerobióza MeSH
• genetické vektory genetika   MeSH
• Giardia lamblia cytologie   genetika   MeSH
• hydrolasy genetika   MeSH
• ligandy MeSH
• mitochondrie metabolismus   MeSH
• molekulární zobrazování metody   MeSH
• organely metabolismus   MeSH
• protozoální proteiny genetika   MeSH
• rekombinantní fúzní proteiny genetika   MeSH
• reportérové geny genetika   MeSH
• Trichomonas vaginalis cytologie   genetika   MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• ligandy MeSH
• protozoální proteiny MeSH
• rekombinantní fúzní proteiny MeSH

Mitochondria are archetypal organelles of endosymbiotic origin in eukaryotic cells. Some unicellular eukaryotes (protists) were considered to be primarily amitochondrial organisms that diverged from the eukaryotic lineage before the acquisition of the premitochondrial endosymbiont, but their amitochondrial status was recently challenged by the discovery of mitochondria-like double membrane-bound organelles called mitosomes. Here, we report that proteins targeted into mitosomes of Giardia intestinalis have targeting signals necessary and sufficient to be recognized by the mitosomal protein import machinery. Expression of these mitosomal proteins in Trichomonas vaginalis results in targeting to hydrogenosomes, a hydrogen-producing form of mitochondria. We identify, in Giardia and Trichomonas, proteins related to the component of the translocase in the inner membrane from mitochondria and the processing peptidase. A shared mode of protein targeting supports the hypothesis that mitosomes, hydrogenosomes, and mitochondria represent different forms of the same fundamental organelle having evolved under distinct selection pressures.

• MeSH
• aktivní transport MeSH
• ferredoxiny genetika   metabolismus   MeSH
• Giardia lamblia genetika   metabolismus   ultrastruktura   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• organely metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• proteiny obsahující železo a síru genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• Trichomonas vaginalis genetika   metabolismus   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• srovnávací studie MeSH
• Názvy látek
• ferredoxiny MeSH
• proteiny obsahující železo a síru MeSH
• protozoální proteiny MeSH
• rekombinantní proteiny MeSH

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.

• MeSH
• biotinylace MeSH
• Escherichia coli enzymologie   MeSH
• frakcionace buněk MeSH
• Giardia lamblia chemie   cytologie   metabolismus   MeSH
• giardiáza parazitologie   MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• ligasy tvořící vazby C-N metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• organely chemie   metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• protozoální proteiny analýza   izolace a purifikace   metabolismus   MeSH
• represorové proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• birA protein, E coli MeSH Prohlížeč
• ligasy tvořící vazby C-N MeSH
• proteiny z Escherichia coli MeSH
• protozoální proteiny MeSH
• represorové proteiny MeSH

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.

• MeSH
• biologická evoluce MeSH
• dynaminy metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• Giardia lamblia cytologie   metabolismus   MeSH
• interfáze MeSH
• koenzym A-ligasy metabolismus   MeSH
• mitochondriální dynamika * MeSH
• mitochondrie metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dynaminy MeSH
• koenzym A-ligasy MeSH
• Long-Chain-Fatty-Acid-CoA Ligase MeSH

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.

• MeSH
• anaerobióza MeSH
• Giardia lamblia enzymologie   MeSH
• mitochondrie enzymologie   MeSH
• molekulární evoluce * MeSH
• sekvence aminokyselin MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transportní proteiny mitochondriální membrány MeSH

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.

• MeSH
• cytoplazma MeSH
• cytosol metabolismus   MeSH
• Giardia lamblia genetika   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• proteiny obsahující železo a síru metabolismus   MeSH
• síra metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteiny obsahující železo a síru MeSH
• síra MeSH
• železo MeSH

• Publikační typ
• časopisecké články MeSH

BACKGROUND: The presence of mitochondria is a distinguishing feature between prokaryotic and eukaryotic cells. It is currently accepted that the evolutionary origin of mitochondria coincided with the formation of eukaryotes and from that point control of mitochondrial inheritance was required. Yet, the way the mitochondrial presence has been maintained throughout the eukaryotic cell cycle remains a matter of study. Eukaryotes control mitochondrial inheritance mainly due to the presence of the genetic component; still only little is known about the segregation of mitochondria to daughter cells during cell division. Additionally, anaerobic eukaryotic microbes evolved a variety of genomeless mitochondria-related organelles (MROs), which could be theoretically assembled de novo, providing a distinct mechanistic basis for maintenance of stable mitochondrial numbers. Here, we approach this problem by studying the structure and inheritance of the protist Giardia intestinalis MROs known as mitosomes. RESULTS: We combined 2D stimulated emission depletion (STED) microscopy and focused ion beam scanning electron microscopy (FIB/SEM) to show that mitosomes exhibit internal segmentation and conserved asymmetric structure. From a total of about forty mitosomes, a small, privileged population is harnessed to the flagellar apparatus, and their life cycle is coordinated with the maturation cycle of G. intestinalis flagella. The orchestration of mitosomal inheritance with the flagellar maturation cycle is mediated by a microtubular connecting fiber, which physically links the privileged mitosomes to both axonemes of the oldest flagella pair and guarantees faithful segregation of the mitosomes into the daughter cells. CONCLUSION: Inheritance of privileged Giardia mitosomes is coupled to the flagellar maturation cycle. We propose that the flagellar system controls segregation of mitochondrial organelles also in other members of this supergroup (Metamonada) of eukaryotes and perhaps reflects the original strategy of early eukaryotic cells to maintain this key organelle before mitochondrial fusion-fission dynamics cycle as observed in Metazoa was established.

• Klíčová slova
• Cell cycle, Cytoskeleton, Flagellum, Giardia, Mitochondrial division, Mitochondrial inheritance, Mitosomes, Protist, mitochondrial evolution,
• MeSH
• databáze genetické MeSH
• Giardia lamblia * genetika   MeSH
• mitochondriální dynamika MeSH
• mitochondrie genetika   MeSH
• organely MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The origin of protein import was a key step in the endosymbiotic acquisition of mitochondria. Though the main translocon of the mitochondrial outer membrane, TOM40, is ubiquitous among organelles of mitochondrial ancestry, the transit peptides, or N-terminal targeting sequences (NTSs), recognised by the TOM complex, are not. To better understand the nature of evolutionary conservation in mitochondrial protein import, we investigated the targeting behavior of Trichomonas vaginalis hydrogenosomal proteins in Saccharomyces cerevisiae and vice versa. Hydrogenosomes import yeast mitochondrial proteins even in the absence of their native NTSs, but do not import yeast cytosolic proteins. Conversely, yeast mitochondria import hydrogenosomal proteins with and without their short NTSs. Conservation of an NTS-independent mitochondrial import route from excavates to opisthokonts indicates its presence in the eukaryote common ancestor. Mitochondrial protein import is known to entail electrophoresis of positively charged NTSs across the electrochemical gradient of the inner mitochondrial membrane. Our present findings indicate that mitochondrial transit peptides, which readily arise from random sequences, were initially selected as a signal for charge-dependent protein targeting specifically to the mitochondrial matrix. Evolutionary loss of the electron transport chain in hydrogenosomes and mitosomes lifted the selective constraints that maintain positive charge in NTSs, allowing first the NTS charge, and subsequently the NTS itself, to be lost. This resulted in NTS-independent matrix targeting, which is conserved across the evolutionary divide separating trichomonads and yeast, and which we propose is the ancestral state of mitochondrial protein import.

• Klíčová slova
• TOM/TIM, hydrogenosomes, mitochondria, mitosomes, protein import,
• MeSH
• mitochondriální proteiny chemie   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce * MeSH
• proteiny - lokalizační signály * MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• transport proteinů MeSH
• Trichomonas vaginalis metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteiny - lokalizační signály * MeSH
• Saccharomyces cerevisiae - proteiny MeSH