Role of a SNARE protein in the biogenesis of Giardia intestinalis mitosomes.

Většina membránových fúzi probíhajících v eukaryotické buňce je zajišťována SNARE proteiny. Pomocí nich probíhá fúze napříč veškerými endocytickými a sekretorickými drahami, což souvisí s jejich specifickou lokalizací v příslušných buněčných kompartmentech. Stranou tohoto mechanismu stojí pouze mitochondrie a plastidy, jejichž fúze je zajišťována specifickými proteinovými mašineriemi. V této práci jsou shrnuty dosavadní poznatky týkající se membránové fúze zajišťované SNARE proteiny a fúze vnější i vnitřní mitochondriální membrány. Důraz je zde kladen na situaci u bičíkatého parazitického prvoka Giardia intestinalis, kde byla navrhnuta unikátní lokalizace typického SNARE proteinu GiSec20 do redukovaných mitochondrií - mitosomů. Tento protein je navíc esenciální pro přežívání trofozoitů G. intestinalis. V této práci jsme ukázali, že mitosomální lokalizace GiSec20 je dosaženo pouze při episomální expresi protenu, zatímco za fyziologických podmínek je protein lokalizován do endoplazmatického retikula, jako je tomu u ostatních eukaryot. Pomocí GFP tagu se nám podařilo lépe charakterizovat targetovací signál, který se ukázal být přítomen v transmembránové doméně proteinu a který byl dostatečný pro targetování proteinu do mitosomů G. intestinalis, respektive mitochondrie S. cerevisiae. Mitosomální...SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

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

Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology

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

Biogenesis of Giardia intestinalis mitosomes

7 ABSTRACT Mitochondria of opisthokonts undergo permanent fusion and fission throughout the cell cycle. Keeping these two processes in balance is vital for various aspects of mitochondrial and cellular homeostasis. Both mitochondrial fusion and division mechanisms are controlled by highly conserved dynamin-related GTPases that are present in all kingdoms of life. The aspects of mitochondrial dynamics outside the opisthokonts is, however, almost completely unexplored phenomenon. In our work, we introduced a tool for live imaging of the reduced forms of mitochondria into model organisms Giardia intestinalis and Trichomonas vaginalis, anaerobic protist parasites from the Excavata supergroup of Eukaryotes. Using this technique, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, of G. intestinalis. The division of mitosomes is restricted to Giardia mitosis and is absolutely synchronized with the process. The synchrony of the nuclear and the mitosomal division persists also during the encystation of the parasite. Surprisingly, the sole dynamin-related protein of the parasite seems not to be involved in mitosomal division. However, throughout the cell cycle mitosomes associate with the..

Biogenesis of mitochondria and related organelles.

Department of ParasitologyKatedra parazitologieFaculty of SciencePřírodovědecká fakult

Role of a SNARE protein in the biogenesis of Giardia intestinalis mitosomes.

SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information..

Biogenesis of Giardia intestinalis mitosomes

7 ABSTRACT Mitochondria of opisthokonts undergo permanent fusion and fission throughout the cell cycle. Keeping these two processes in balance is vital for various aspects of mitochondrial and cellular homeostasis. Both mitochondrial fusion and division mechanisms are controlled by highly conserved dynamin-related GTPases that are present in all kingdoms of life. The aspects of mitochondrial dynamics outside the opisthokonts is, however, almost completely unexplored phenomenon. In our work, we introduced a tool for live imaging of the reduced forms of mitochondria into model organisms Giardia intestinalis and Trichomonas vaginalis, anaerobic protist parasites from the Excavata supergroup of Eukaryotes. Using this technique, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, of G. intestinalis. The division of mitosomes is restricted to Giardia mitosis and is absolutely synchronized with the process. The synchrony of the nuclear and the mitosomal division persists also during the encystation of the parasite. Surprisingly, the sole dynamin-related protein of the parasite seems not to be involved in mitosomal division. However, throughout the cell cycle mitosomes associate with the..

Biogeneze mitosomů Giardia intestinalis

8 ABSTRAKT Mitochondrie opisthokont neustále fúzují a dělí se v průběhu celého buněčného cyklu. Udržení těchto dvou procesů v rovnováze je pro buňku zásadní. Mitochondriální fúze i dělení jsou řízeny dynaminovými GTPázami, které jsou konzervovány napříč všemi organismy. Jak mitochondriální fúze a dělení probíhá mimo zmíněnou skupinu organismů téměř není známo. V naší práci jsme se zabývali zavedením fluorescenčního značení pro live imaging do organismů G. intestinalis a T. vaginalis, jednobuněčných parazitů ze skupiny Excavata. Pomocí této metody jsme poté zkoumali dynamiku mitosomů, nejjednodušších forem mitochondrií, u G. intestinalis. Zjistili jsme, že dělení mitosomů probíhá během mitozy, se kterou je absolutně synchronizováno, a že ke stejné synchronizaci dochází také během encystace parazita. Dále jsme objevili, že během buněčného cyklu jsou mitosomy spojené s endoplasmatickým retikulem, nicméně charakter tohoto spojení není znám, jelikož genom Giardie nekóduje žádný ze známých proteinů zodpovědných za zprostředkování tohoto kontaktu. Prozatím jediným proteinem nalezeným v místech kontaktu mitosomů a endoplasmatického retikula je acyl-CoA syntetáza 4, enzym biosyntézy lipidů. Také jsme se zabývali hledáním dalších potenciálních mitosomálních ...7 ABSTRACT Mitochondria of opisthokonts undergo permanent fusion and fission throughout the cell cycle. Keeping these two processes in balance is vital for various aspects of mitochondrial and cellular homeostasis. Both mitochondrial fusion and division mechanisms are controlled by highly conserved dynamin-related GTPases that are present in all kingdoms of life. The aspects of mitochondrial dynamics outside the opisthokonts is, however, almost completely unexplored phenomenon. In our work, we introduced a tool for live imaging of the reduced forms of mitochondria into model organisms Giardia intestinalis and Trichomonas vaginalis, anaerobic protist parasites from the Excavata supergroup of Eukaryotes. Using this technique, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, of G. intestinalis. The division of mitosomes is restricted to Giardia mitosis and is absolutely synchronized with the process. The synchrony of the nuclear and the mitosomal division persists also during the encystation of the parasite. Surprisingly, the sole dynamin-related protein of the parasite seems not to be involved in mitosomal division. However, throughout the cell cycle mitosomes associate with the...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Role of a SNARE protein in the biogenesis of Giardia intestinalis mitosomes.

SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information..

Mitochondrial dynamics in parasitic protists.

The shape and number of mitochondria respond to the metabolic needs during the cell cycle of the eukaryotic cell. In the best-studied model systems of animals and fungi, the cells contain many mitochondria, each carrying its own nucleoid. The organelles, however, mostly exist as a dynamic network, which undergoes constant cycles of division and fusion. These mitochondrial dynamics are driven by intricate protein machineries centered around dynamin-related proteins (DRPs). Here, we review recent advances on the dynamics of mitochondria and mitochondrion-related organelles (MROs) of parasitic protists. In contrast to animals and fungi, many parasitic protists from groups of Apicomplexa or Kinetoplastida carry only a single mitochondrion with a single nucleoid. In these groups, mitochondrial division is strictly coupled to the cell cycle, and the morphology of the organelle responds to the cell differentiation during the parasite life cycle. On the other hand, anaerobic parasitic protists such as Giardia, Entamoeba, and Trichomonas contain multiple MROs that have lost their organellar genomes. We discuss the function of DRPs, the occurrence of mitochondrial fusion, and mitophagy in the parasitic protists from the perspective of eukaryote evolution