Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei

Käser, Sandro; Willemin, Mathilde Stéphanie; Schnarwiler, Felix; Schimanski, Bernd; Poveda-Huertes, Daniel; Oeljeklaus, Silke; Haenni, Beat; Zuber, Benoît; Warscheid, Bettina; Meisinger, Chris; Schneider, André

doi:10.1371/journal.ppat.1006808

Cited by 23 publications

(34 citation statements)

References 59 publications

(92 reference statements)

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“…The current model of the TAC contains 13 components four of which (p197, BBA4, Mab22 and TAC65) are localised to the exclusion zone filaments (Gheiratmand et al 2013;Bonhivers et al 2008;Käser et al 2016) . Six components are in the differentiated mitochondrial membranes, four in the outer mitochondrial membrane TAC60, TAC42, TAC40 and pATOM36 (Schnarwiler et al 2014;Käser et al 2016Käser et al , 2017 and two components are likely associated with the inner mitochondrial membrane (p166, AEP1, (Zhao et al 2008;Ochsenreiter and Hajduk 2006) ). TAC102 is a protein of the unilateral filaments and the most proximal component to the kDNA (Trikin et al 2016;Hoffmann, Jakob, and Ochsenreiter 2016) .…”

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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“…Polyclonal antibodies against TbMic10‐1 (used in a 1:1,000 dilution) and TbMic34 (diluted 1:2,500 for immunoblots) were obtained upon immunization of rabbits with a synthetic peptide (LRKGFRGSTGVSDRESSGS) or recombinant full‐length protein expressed in E. coli respectively. All other antibodies are described in detail (Käser et al , ).…”

Section: Methodsmentioning

confidence: 99%

The highly diverged trypanosomal MICOS complex is organized in a nonessential integral membrane and an essential peripheral module

Eichenberger¹,

Oeljeklaus

Bruggisser³

et al. 2019

Molecular Microbiology

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Summary The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10‐like and a Mic60‐like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome‐wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co‐regulation of TbMic10‐1, TbMic10‐2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space‐localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10‐containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane‐integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane‐peripheral one consisting of four kinetoplastid‐specific subunits, that is required for import of intermembrane space proteins.

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“…The mitochondrial protein TAC102 and other TAC components can be partially solubilized by digitonin extractions as shown in earlier studies [30,34,36] . At a concentration of 0.025% digitonin cytoplasmic components can be separated from crude organellar structures containing the mitochondrial organelle.…”

Section: Tap110 Is Insoluble In Kdna Containing Cellsmentioning

confidence: 99%

“…The closest interactor of TAC102 is the transmembrane domain containing protein p166 that is localized at the inner mitochondrial membrane [32] . Three outer mitochondrial membrane components of the TAC (TAC40, TAC42 and TAC60, [33,34] ) as well as two…”

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Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 inTrypanosoma brucei

Amodeo

Kalichava

Fradera-Sola

et al. 2020

Preprint

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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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Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei

Cited by 23 publications

References 59 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

The highly diverged trypanosomal MICOS complex is organized in a nonessential integral membrane and an essential peripheral module

Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 inTrypanosoma brucei

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