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Tubular organization of the TAC and functional role of TAC53 – TAC42 RNAi data

ABSTRACT: 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 TACcomponent 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 the TAC–kDNA 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.

INSTRUMENT(S):

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Julian Bender

LAB HEAD: Bettina Warscheid

PROVIDER: PXD063233 | Pride | 2025-08-04

REPOSITORIES: Pride

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			Action	DRS
	ELITE-RSLC019640.raw	Raw
	ELITE-RSLC019642.raw	Raw
	ELITE-RSLC019645.raw	Raw
	MaxQ_txt_TAC42.zip	Other

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Tubular organization of the TAC and functional role of TAC53 – p197 RNAi data

Project description: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 TACcomponent 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 the TAC–kDNA 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.

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

Project description: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.

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Project description: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 the TAC-kDNA 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.

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