Nejvíce citovaný článek - PubMed ID 27926928
Myeloid leukemia factor 1 (Mlf1) was identified as a proto-oncoprotein that affects hematopoietic differentiation in humans. However, its cellular function remains elusive, spanning roles from cell cycle regulation to modulation of protein aggregate formation and participation in ciliogenesis. Given that structurally conserved homologs of Mlf1 can be found across the eukaryotic tree of life, we decided to characterize its cellular role underlying this phenotypic pleiotropy. Using a model of the unicellular eukaryote Giardia intestinalis, we demonstrate that its Mlf1 homolog (GiMlf) mainly localizes to two types of cytosolic foci: microtubular structures, where it interacts with Hsp40, and ubiquitin-rich, membraneless compartments, found adjacent to mitochondrion-related organelles known as mitosomes, containing the 26S proteasome regulatory subunit 4. Upon cellular stress, GiMlf either relocates to the affected compartment or disperses across the cytoplasm, subsequently accumulating into enlarged foci during the recovery phase. In vitro assays suggest that GiMlf can be recruited to membranes through its affinity for signaling phospholipids. Importantly, cytosolic foci diminish in the gimlf knockout strain, which exhibits extensive proteomic changes indicative of compromised proteostasis. Consistent with data from other cellular systems, we propose that Mlf acts in the response to proteotoxic stress by mediating the formation of function-specific foci for protein folding and degradation.
- MeSH
- Giardia lamblia * metabolismus MeSH
- lidé MeSH
- proteolýza * MeSH
- protozoální proteiny * metabolismus genetika MeSH
- sbalování proteinů * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- protozoální proteiny * MeSH
Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. In addition to IscA2, Nfu1 and Grx5 we identified a novel BolA1 homologue in G. intestinalis mitosomes. It specifically interacts with Grx5 and according to the high-affinity pulldown also with other core mitosomal components. Using CRISPR/Cas9 we were able to establish full bolA1 knock out, the first cell line lacking a mitosomal protein. Despite the ISC pathway being the only metabolic role of the mitosome no significant changes in the mitosome biology could be observed as neither the number of the mitosomes or their capability to form [2Fe-2S] clusters in vitro was affected. We failed to identify natural client proteins that would require the [2Fe-2S] or [4Fe-4S] cluster within the mitosomes, with the exception of [2Fe-2S] ferredoxin, which is itself part of the ISC pathway. The overall uptake of iron into the cellular proteins remained unchanged as also observed for the grx5 knock out cell line. The pull-downs of all late ISC components were used to build the interactome of the pathway showing specific position of IscA2 due to its interaction with the outer mitosomal membrane proteins. Finally, the comparative analysis across Metamonada species suggested that the adaptation of the late ISC pathway identified in G. intestinalis occurred early in the evolution of this supergroup of eukaryotes.
- MeSH
- anaerobióza MeSH
- Giardia lamblia * genetika metabolismus MeSH
- lidé MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- proteiny obsahující železo a síru * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mitochondriální proteiny MeSH
- proteiny obsahující železo a síru * 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
BACKGROUND: Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia's pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. RESULTS: We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. CONCLUSIONS: Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.
- Klíčová slova
- ESCRT, Endomembrane, Evolutionary Cell Biology, Excavata, Giardia, PV, Parasitism,
- MeSH
- biologická evoluce MeSH
- endozomální třídící komplexy pro transport * genetika metabolismus MeSH
- endozomy metabolismus MeSH
- Giardia lamblia * genetika metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- endozomální třídící komplexy pro transport * MeSH
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.
The discovery that the protist Monocercomonoides exilis completely lacks mitochondria demonstrates that these organelles are not absolutely essential to eukaryotic cells. However, the degree to which the metabolism and cellular systems of this organism have adapted to the loss of mitochondria is unknown. Here, we report an extensive analysis of the M. exilis genome to address this question. Unexpectedly, we find that M. exilis genome structure and content is similar in complexity to other eukaryotes and less "reduced" than genomes of some other protists from the Metamonada group to which it belongs. Furthermore, the predicted cytoskeletal systems, the organization of endomembrane systems, and biosynthetic pathways also display canonical eukaryotic complexity. The only apparent preadaptation that permitted the loss of mitochondria was the acquisition of the SUF system for Fe-S cluster assembly and the loss of glycine cleavage system. Changes in other systems, including in amino acid metabolism and oxidative stress response, were coincident with the loss of mitochondria but are likely adaptations to the microaerophilic and endobiotic niche rather than the mitochondrial loss per se. Apart from the lack of mitochondria and peroxisomes, we show that M. exilis is a fully elaborated eukaryotic cell that is a promising model system in which eukaryotic cell biology can be investigated in the absence of mitochondria.
- Klíčová slova
- Monocercomonoides, oxymonads, protist genomics, amitochondrial eukaryote, cell biology,
- MeSH
- genom protozoální * MeSH
- intracelulární membrány * MeSH
- introny MeSH
- mikrofilamenta MeSH
- mitochondriální dynamika MeSH
- Oxymonadida enzymologie genetika ultrastruktura MeSH
- proteom MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteom 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
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.
- Klíčová slova
- Evolution, Excavata, Fe–S cluster, Mitochondria,
- MeSH
- Eukaryota cytologie metabolismus MeSH
- mitochondrie metabolismus MeSH
- proteiny obsahující železo a síru metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- proteiny obsahující železo a síru MeSH
- železo 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
- mastná kyselina s dlouhým řetězcem-CoA-ligasa MeSH
- mitochondriální dynamika * MeSH
- mitochondrie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- dynaminy MeSH
- koenzym A-ligasy MeSH
- mastná kyselina s dlouhým řetězcem-CoA-ligasa MeSH
