Jetishi, C.; Aeschlimann, S.; Schimanski, B.; Kaeser, S.; Mullner, R.; Oeljeklaus, S.; Akiyoshi, B.; Warscheid, B.; Butter, F.; Schneider, A.; Ochsenreiter, T.

The Tripartite Attachment Complex (TAC) is essential for mitochondrial DNA (kDNA) segregation in Trypanosoma brucei, providing a physical link between the flagellar basal body and the mitochondrial genome. Although the TAC\'s hierarchical assembly and linear organization have been extensively studied, much remains to be discovered regarding its complete architecture and composition, for instance, our identification of a new TAC component underscores these knowledge gaps. Here, we use a combination of proteomics, RNA interference (RNAi), and Ultrastructure Expansion Microscopy (U-ExM) to characterize the TAC at high resolution and identify a novel component, TAC53 (Tb927.2.6100). Depletion of TAC53 in both procyclic and bloodstream forms results in kDNA missegregation and loss, a characteristic feature of TAC dysfunction. TAC53 localizes to the kDNA in a cell cycle-dependent manner and represents the most kDNA-proximal TAC component identified to date. U-ExM reveals a previously unrecognized tubular architecture of the TAC, with two distinct TAC structures per kDNA disc, suggesting a mechanism for precise kDNA alignment and segregation. Moreover, immunoprecipitation and imaging analyses indicate that TAC53 interacts with known TAC-associated proteins HMG44, KAP68, and KAP3, forming a network at TAC-kDNA the interface. These findings redefine our understanding of TAC architecture and function and identify TAC53 as a key structural component anchoring the mitochondrial genome in T. brucei.