Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC. Author summaryTrypanosoma brucei is a single celled eukaryote that is only distantly related to most other model organisms. It is important to study this group in order to understand which parts of their biology are conserved and which have diverged throughout evolution. T. brucei cells-like most eukaryotes-contain mitochondria. Trypanosomes however, only harbor one mitochondrion with a singular mitochondrial genome per cell. It has been shown that the mitochondrial genome in these organisms is physically linked to the base of the flagellum and that this linkage, which has been named Tripartite Attachment Complex (TAC), is required for proper distribution of the mitochondrial genome during cell division. In the current study we identify and characterize two novel components of a distinct complex involved in connecting the TAC to the mitochondrial DNA.