Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 inTrypanosoma brucei

Abstract

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...

Characterization of two novel proteins involved in mitochondrial DNA anchoring

“…We recently showed that TAC102 as well as TAP110 remain at the flagellum even in dyskinetoplastic cells, supporting the model that TAC assembly is independent of kDNA (Amodeo et al, 2020;Hoffmann et al, 2018). To test whether this is also the case for TbmtHMG44 and Tb927.11.16120, we performed flagellar extractions in presence and absence of an endonulcease (DNase I).…”

“…We identified 1523 proteins and among the top 50 enriched proteins, 21 were predicted/annotated to be mitochondrion or axonem associated. This included six already characterized TAC components: TAC102, TAP110, p166, TAC60, TAC65, and TAC40 (Amodeo et al, 2020;Käser et al, 2016;Käser et al, 2017;Schnarwiler et al, 2014;Trikin et al, 2016;Zhao et al, 2008). Also among the top 50 enriched proteins we detected the two very basic, potentially essential proteins, Tb927.9.5020 and Tb927.11.16120 that were predicted to be kinetoplast and mitochondrially localized, respectively (Table S1).…”

“…Furthermore, the abundance of four kDNA associated proteins Tb927.11.6660, TbmtHMG44, Tb927.10.8980 and Tb927.11.16120 was affected by TAC102 depletion ( Figure 1B). Tb927.2.6100 was previously described as a kDNA associated factor important for kDNA maintenance (Beck et al, 2013) while Tb927.11.6660 was recently identified as an interactor of TAC102 (Amodeo et al, 2020). Mass spectrometry also revealed the presence of 25 mitochondrial DNA replication factors in the DNase treated flagellar extracts (Table S2).…”

“…We now know that the acidic TAC/ULF protein p166 is associated with the inner mitochondrial membrane and directly interacts with the basic more kDNA proximal ULF protein TAC102 (Baudouin et al, 2019;Trikin et al, 2016;Zhao et al, 2008). Towards the kDNA TAC102 is connected to several proteins including the recently characterized basic protein TAP110 (pI 8, (Amodeo et al, 2020)) which is closer to the kDNA than TAC102. We have no evidence that TAC102 binds directly to DNA and based on recent U-ExM data there is clearly a gap between TAC102 and the DNA supporting the model that there are additional proteins between TAC102 and the kDNA (Amodeo et al, 2020).…”

“…The DM harbor four proteins of the TAC in the outer mitochondrial membrane (TAC60, TAC42, TAC40 and pATOM36, (Käser et al, 2016;Käser et al, 2017;Schnarwiler et al, 2014)) while one component is associated with the inner mitochondrial membrane (p166, (Ochsenreiter and Hajduk, 2006;Zhao et al, 2008)). The only known protein components of the ULF are TAC102 (Trikin et al, 2016) and TAP110 (Amodeo et al, 2020). While TAP110 is proximal to the kDNA when compared with TAC102, it does not seem to be essential for kDNA maintenance as other TAC proteins are (Schneider and Ochsenreiter, 2018).…”