Characterization of the novel mitochondrial genome replication factor MiRF172 in <i>Trypanosoma brucei</i>

Amodeo, Simona; Jakob, Martin; Ochsenreiter, Torsten

doi:10.1242/jcs.211730

Cited by 14 publications

(18 citation statements)

References 57 publications

(97 reference statements)

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“…To analyze the localization of TAP110, TAC102, and basal body proteins, we used immunofluorescence analysis as described previously [53] . Primary and secondary antibodies were diluted as follows: polyclonal…”

Section: Immunofluorescence Analysismentioning

confidence: 99%

“…To analyze presence and/or abundance of a protein of interest in whole cell lysates and fractions from digitonin and flagellar extraction we used western blot analysis. Samples were prepared as described previously [53] . Approximately 5×10 6 cells were loaded per lane and SDS PAGE and transferred to PVDF membrane.…”

Section: Sds-page and Western Blottingmentioning

confidence: 99%

“…Approximately 5×10 6 cells were loaded per lane and SDS PAGE and transferred to PVDF membrane. blocking and detection of proteins of interest (PTP and HA tagged proteins, EF1alpha and ATOM40) was performed as described previously [53] . Alpha tubulin was detected by the monoclonal mouse anti-alpha tubulin antibody (1:20'000, Sigma) and the rabbit anti-mouse-IgG conjugated to horseradish peroxidase (1:10'000, Dako).…”

Section: Sds-page and Western Blottingmentioning

confidence: 99%

“…To analyze biochemical properties of TAP110 we performed digitonin fractionation as described before [53] . 5x10 6 cell equivalents of each fraction was used for the western blot analysis.…”

Section: Digitonin Fractionationsmentioning

confidence: 99%

“…For flagellar preparation [28] , we performed the experiment as described before [53] . For DNase treatments, second extraction buffer was supplemented with DNaseI (Roche) to a final concentration of 100μg/ml.…”

Section: Flagellar Extractionmentioning

confidence: 99%

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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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Section: Immunofluorescence Analysismentioning

confidence: 99%

Section: Sds-page and Western Blottingmentioning

confidence: 99%

Section: Sds-page and Western Blottingmentioning

confidence: 99%

Section: Digitonin Fractionationsmentioning

confidence: 99%

Section: Flagellar Extractionmentioning

confidence: 99%

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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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Kinetoplast Division Factors in a Trypanosome

Mensa-Wilmot

Hoffman

Wiedeman

et al. 2019

Trends in Parasitology

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Inheritance of the single mitochondrial nucleoid (kinetoplast) in the trypanosome requires numerous proteins many of whose precise roles are unclear. By considering kinetoplast DNA (kDNA) as a template for cleavage into two equal-size networks, we predicted sets of mutant kinetoplasts associated with defects in each of five steps in the kinetoplast cycle. Comparison of these kinetoplasts with those obtained after gene knockdowns enabled assignment of proteins to five classes -kDNA synthesis, site of scission selection, scission, separation, and partitioning. These studies highlight how analysis of mutant kinetoplast phenotypes may be used to predict functional categories of proteins involved in biogenesis of kinetoplasts.

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Importance of Angomonas deanei KAP4 for kDNA arrangement, cell division and maintenance of the host-bacterium relationship

Gonçalves¹,

Catta-Preta²,

Repolês³

et al. 2021

Sci Rep

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Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.

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Characterization of the novel mitochondrial genome replication factor MiRF172 in Trypanosoma brucei

Cited by 14 publications

References 57 publications

Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 inTrypanosoma brucei

Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 inTrypanosoma brucei

Kinetoplast Division Factors in a Trypanosome

Importance of Angomonas deanei KAP4 for kDNA arrangement, cell division and maintenance of the host-bacterium relationship

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