Stettler, Philip (2025). Functional Architecture of the Tripartite Attachment Complex of Trypanosoma brucei. (Thesis). Universität Bern, Bern

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Abstract

Eukaryotic cells are highly complex and contain many organelles. One of the defining features of eukaryotes is the mitochondrion, which is a remnant of the endosymbiotic event that gave rise to this domain of life. This Thesis features mitochondrial genome inheritance systems and highlights the systems found in Trypanosoma brucei, a member of the understudied Discoba supergroup. Mitochondrial genome inheritance has been studied since many years. However, how the replicated mitochondrial genomes are segregated during cell division is not well understood. In human and yeast cells, which are comparatively closely related species classified as Opisthokonta in the supergroup Amorphea, mitochondrial genomes are packed in nucleoids which are trafficked along cytoskeletal structures. Similar principles may apply to the mitochondria of land plants, which belong to the supergroup Archaeplastida, although there are also clear differences. Outside of these two supergroups, the mitochondrial genome segregation system of T. brucei is the only one that has been thoroughly investigated. T. brucei has a single mitochondrion with a genome that is condensed into a single nucleoid, known as the kinetoplast. The kinetoplast is segregated during cell division by basal body movements. This is made possible by a protein complex that connects the two structures: the tripartite attachment complex (TAC). The TAC consists of at least nine subunits, each present in several hundred to a few thousand copies. The role, localization, and direct interaction partners of the nine TAC subunits are well known. This Thesis contains two studies focusing on the TAC. The first one highlights the contact site between the outer and inner mitochondrial membranes of the TAC and the second focuses on the assembly of the mitochondrial outer membrane TAC module. A third study, which is not related to the TAC, reports the discovery of a novel pathway regulating mitochondrial DNA replication. The first study focuses on TAC60 and p166, two TAC subunits located in the outer and inner mitochondrial membranes, respectively. Previous studies have shown that these proteins interact directly to form a unique and permanent contact site between the outer and inner mitochondrial membranes that is essential for TAC function. Our goal was to characterize this interaction at a molecular level. We identified the interaction domains down to the amino acid level. Our results suggest that hydrophobic interfaces are at the core of this membrane contact site. This was an unexpected finding, as the interaction domains in both proteins contain well conserved charged amino acids. In the second study we investigated the assembly of the mitochondrial outer membrane TAC module. This module is the most complex of the three TAC modules. It contains five subunits: pATOM36, TAC40, TAC42, TAC60, and TAC65. Although the TAC assembles de novo and unidirectionally from the basal body towards the kinetoplast, the subunits of the mitochondrial outer membrane TAC module do not strictly follow this order. In this study, we identified four detergent-soluble assembly intermediates, which can be grouped into two classes. One class contains an oligomeric TAC40 subcomplex, as well as two more complicated TAC40-, TAC42-, TAC60-containing subcomplexes which likely originate from a shared assembly pathway. The second class contains a single subcomplex containing pATOM36 and TAC65. Our results suggest that the largest assembly intermediate from the first class merges with the assembly intermediate of the second class to form the mitochondrial outer membrane TAC module. In addition, we show that the N-terminal domain of TAC60 is essential for this last step. The third study focuses on kinetoplast DNA maintenance and replication and is unrelated to the TAC. The kinetoplast is an intricate DNA network containing two classes of circular DNA molecules, maxicircles and minicircles. We identified a novel pathway relevant for maxicircle level regulation in T. brucei, comprising the three proteins MaRF11, TbPam16, and TbPam18. Notably, TbPam16 and TbPam18 are orthologs of proteins involved in mitochondrial protein import in other lineages. Our findings revealed that in trypanosomes, these proteins have acquired lineage-specific functions. Together with the newly discovered MaRF11 protein, they contribute to the regulation of maxicircle replication by yet unknown, life cycle stage-specific pathways. Interestingly, TbPam16 and TbPam18 are mitochondrial inner membrane proteins and this localization is essential for their function in maxicircle maintenance and/or replication.

Item Type:	Thesis
Dissertation Type:	Cumulative
Date of Defense:	21 August 2025
Subjects:	500 Science > 540 Chemistry 600 Technology > 610 Medicine & health
Institute / Center:	08 Faculty of Science
Depositing User:	Hammer Igor
Date Deposited:	30 Sep 2025 15:33
Last Modified:	09 Oct 2025 02:05
URI:	https://boristheses.unibe.ch/id/eprint/6756

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