Failure is not an option – mitochondrial genome segregation in trypanosomes

Schneider, André; Ochsenreiter, Torsten

doi:10.1242/jcs.221820

Cited by 59 publications

(84 citation statements)

References 87 publications

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“…A number of additional components that display multiple localisations including in the TAC, like the E2 subunit of the α-ketoglutarate dehydrogenase and the tubulin-binding cofactor C protein, have been identified (Sykes and Hajduk 2013;André et al 2013) . The assembly of the TAC occurs de novo from the base of the flagellum towards the kDNA in a hierarchical manner such that kDNA proximal components depend on the proper assembly of the kDNA distal components (Hoffmann et al 2018;Schneider and Ochsenreiter 2018) . In order to identify novel interactors of TAC102, we used three different approaches with TAC102 as the bait: (i) proximity-dependent biotin identification (BioID), (ii) immunoprecipitation with a monoclonal TAC102 antibody and (iii) yeast two-hybrid.…”

Section: Introductionmentioning

confidence: 99%

Discovery of new mitochondrial DNA segregation machinery components inTrypanosoma brucei: a comparison of three approaches

Hcm

Pfeiffer

Ochsenreiter

2019

Preprint

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Trypanosoma brucei is a single celled eukaryotic parasite and the causative agent of human African sleeping sickness and Nagana in cattle. Aside from its medical relevance T. brucei has also been key to the discovery of several general biological principles including GPI-anchoring, RNA-editing and trans-splicing. The parasite contains a single mitochondrial organelle with a singular genome. Recent studies have identified several molecular components of the mitochondrial genome segregation machinery (tripartite attachment complex, TAC), which connects the basal body of the flagellum to the mitochondrial DNA of T. brucei . The TAC component in closest proximity to the mitochondrial DNA is TAC102.Here we apply and compare three different approaches (proximity labeling, Immunoprecipitation and yeast two-hybrid) to identify novel interactors of TAC102 and subsequently verify their localisation. Furthermore, we establish the direct interaction of TAC102 and p166 in the unilateral filaments of the TAC.

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Section: Introductionmentioning

confidence: 99%

Discovery of new mitochondrial DNA segregation machinery components inTrypanosoma brucei: a comparison of three approaches

Hcm

Pfeiffer

Ochsenreiter

2019

Preprint

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“…This might suggest, that while most of the TAC machinery is conserved in all Kinetoplastea, the components in proximity to the DNA have adapted to the different kDNA conformations or replication mechanisms. We previously described a number of criteria for proteins of the TAC [29] . First, a TAC component is localized between the kDNA disc and the basal body of the flagellum.…”

Section: Discussionmentioning

confidence: 99%

“…The TAC consists of (i) the exclusion zone filaments, a region between the basal bodies and the outer mitochondrial membrane that is devoid of ribosomes, (ii) the differentiated mitochondrial membranes and (iii) the unilateral filaments that connect the inner mitochondrial membrane to the kDNA [28] . Several proteins of this structure have been characterized [23,29] . TAC102 is the kDNA most proximal TAC component known.…”

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Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 inTrypanosoma brucei

Amodeo

Kalichava

Fradera-Sola

et al. 2020

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KeywordskDNA, mitochondrial genome segregation machinery, tripartite attachment complex (TAC), T AC A ssociated P rotein 110 , Trypanosoma brucei, ultrastructure expansion microscopy (U-ExM) Summary StatementTAP110 is a novel mitochondrial genome segregation factor in Trypanosoma brucei that associates with the previously described TAC component TAC102. Ultrastructure expansion microscopy reveals its proximal position to the kDNA. AbstractProper mitochondrial genome inheritance is key for eukaryotic cell survival, however little is known about the molecular mechanism controlling this process. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome aka kinetoplast DNA (kDNA). kDNA segregation requires anchoring of the genome to the basal body via the tripartite attachment complex (TAC). Several components of the TAC as well as their assembly have been described, it however remains elusive how the TAC connects to the kDNA.Here, we characterize the TAC associated protein TAP110 and for the first time use ultrastructure expansion microscopy in trypanosomes to reveal that TAP110 is the currently most proximal kDNA segregation factor.The kDNA proximal positioning is also supported by RNAi depletion of TAC102, which leads to loss of TAP110 at the TAC. Overexpression of TAP110 leads to expression level changes of several mitochondrial proteins and a delay in the separation of the replicated kDNA networks. In contrast to other kDNA segregation factors TAP110 remains only partially attached to the flagellum after DNAse and detergent treatment and can only be solubilized in dyskinetoplastic cells, suggesting that interaction with the kDNA might be important for stability of the TAC association. Furthermore, we demonstrate that the TAC, but not the kDNA, is required for correct TAP110 localization in vivo and suggest that TAP110 might interact with other proteins to form a >669 kDa complex. Mitochondria are a defining feature of eukaryotic cells. They perform a large number of different functions ranging from catabolic reactions like oxidative phosphorylation [1] to anabolic processes like iron sulfur cluster assembly [2] and calcium homeostasis [3] . The vast majority of the mitochondrial proteins are encoded and expressed from the nuclear genome, while only a small set of proteins, mostly of the oxidative phosphorylation chain are encoded on the organelle's own genome. . In Trypanosoma brucei a parasitic protist the mitochondrial genome is organized in a complex structure named kinetoplast DNA (kDNA). It consists of about 25 large (23 kbp) circular DNA molecules [4] that encode 16 genes of the oxidative phosphorylation chain, two ribosomal proteins [5] and two ribosomal RNAs. Twelve of the mitochondrial genes require posttranscriptional modifications by RNA editing prior to translation on the mitochondrial ribosomes [6-9] . The guide RNAs involved in this process are encoded on minicircles (1 kbp) of which about 5000 are catenated into the kDNA network forming a disc like structure [10] . In that...

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“…Overall there are 5-10´000 minicircles that are catenated with the maxicircles forming the kinetoplast DNA (kDNA) (Shapiro and Englund, 1995;Vickerman, 1977). Currently, more than 30 proteins have been described to be involved in the replication and segregation of the kDNA, while far fewer are required for this process in other eukaryotic systems (Jensen and Englund, 2012;Povelones, 2014;Schneider and Ochsenreiter, 2018). The model for kDNA replication predicts the release of minicircles into the region between the kDNA disc and the inner mitochondrial membrane, named the kinetoflagellar zone (KFZ) .…”

Section: Introductionmentioning

confidence: 99%

Characterization of two novel proteins involved in mitochondrial DNA anchoring

Amodeo

Hoffmann

Fradera-Sola³

et al. 2020

Preprint

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Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Excavates. T. brucei cells harbor a single mitochondrion with a singular mitochondrial genome, that consists of a unique network of thousands of interwoven circular DNA molecule copies and is termed the kinetoplast DNA (kDNA). To ensure proper inheritance of the kDNA to the daughter cells the genome is linked to the basal body, the master organizer of the cell cycle in trypanosomes. The structure connecting the basal body and kDNA is termed the tripartite attachment complex (TAC). Using a combination of proteomics and RNAi (depletomics) we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and Tb927.11.16120 as novel candidates of a structure that connects the TAC to the kDNA. Both proteins localize in the region of the unilateral filaments between TAC102 and the kDNA and depletion of each leads to a strong kDNA loss phenotype. TbmtHMG44 and Tb927.11.16120 stably associate with extracted flagella, even after DNase treatment however they do require the kDNA for initial assembly. Furthermore we demonstrate that recombinant Tb927.11.16120 is a DNA binding protein and thus a promising candidate to link the TAC to the kDNA.

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Failure is not an option – mitochondrial genome segregation in trypanosomes

Cited by 59 publications

References 87 publications

Discovery of new mitochondrial DNA segregation machinery components inTrypanosoma brucei: a comparison of three approaches

Discovery of new mitochondrial DNA segregation machinery components inTrypanosoma brucei: a comparison of three approaches

Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 inTrypanosoma brucei

Characterization of two novel proteins involved in mitochondrial DNA anchoring

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