Biology subjects

Stettler, P.

Publications and source records attributed to Stettler, P..

5 recordsLinked to original sources

bioRxiv2025

Assembly of the mitochondrial outer membrane module of the trypanosomal tripartite attachment complex

The parasitic protozoan Trypanosoma brucei has a single mitochondrial nucleoid, anchored to the basal body of the flagellum via the tripartite attachment complex (TAC). The detergent-insoluble TAC is essential for mitochondrial genome segregation during cytokinesis. The TAC assembles de novo in a directed way from the probasal body towards the kDNA. However, the OM TAC module which is composed of five subunits, has previously been suspected to follow more complicated assembly pathways. Here, we identified four detergent-soluble OM TAC module subcomplexes that we assign to two classes. One class contains an oligomeric TAC40 complex that according to AlphaFold contains 6-8 subunits, as well as two subcomplexes of different sizes comprising TAC40, TAC42, and TAC60. The second class consists of a single complex composed of TAC65 and pATOM36. We show that the two subcomplex classes form independently and accumulate upon impairment of TAC assembly. The expression of an N-terminally truncated TAC60 variant causes the accumulation of the larger TAC40/TAC42/TAC60 complex and blocks completion of OM TAC module assembly. This suggests the following assembly pathway: i) TAC40 oligomerizes, ii) TAC42 and TAC60 bind the TAC40 oligomer forming two discrete larger intermediates, where iii) the larger subcomplex merges with the pATOM36/TAC65 subcomplex subsequently forming the OM TAC module. Author summaryMitochondria are essential for nearly all eukaryotes. This requires proper segregation of the organelles and their genomes to the daughter cells during cell division. For Trypanosomes and its relatives this represents a unique challenge because they have only a single highly compacted mitochondrial nucleoid, containing the bipartite mitochondrial genome, known as the kDNA. To ensure faithful segregation, kDNA replication is tightly coordinated with the nuclear cell cycle and the kDNA is physically connected to the flagellar basal body via the tripartite attachment complex (TAC), which spans both mitochondrial membranes. The TAC consists of at least nine subunits and its biogenesis arguably implies the most extreme lateral sorting event known for any mitochondrion. Five subunits, four integral and one peripheral outer membrane protein, make up the outer TAC module. Using blue native PAGE and RNAi analyses we have identified four soluble subcomplexes that likely represent assembly intermediates of the outer membrane TAC module. These results suggest that the outer membrane TAC module is assembled following a branched pathway that includes an unusual a ring-like intermediate of 6-8 subunits of a trypanosomatid-specific VDAC-like beta-barrel protein.

biochemistry↗

bioRxiv2025

TbTim20 facilitates protein import at a low membrane potential in trypanosomes lacking the mitochondrial genome

Protein import across the mitochondrial inner membrane typically depends on two protein translocases of the inner membrane (TIM) complexes and the membrane potential. The protozoan parasite Trypanosoma brucei, however, has a single, divergent TIM complex. Unlike other trypanosomal TIM subunits, TbTim20 is neither essential for normal growth of insect nor bloodstream forms of T. brucei, leaving its role uncertain. Specific mutations in the {gamma}-subunit of the F1FO-ATPase, such as {gamma}L262P, permit bloodstream form trypanosomes to grow without mitochondrial DNA (kinetoplast or kDNA). Here we show that RNAi-mediated depletion of TbTim20 inhibits growth of this cell line, but only if it lacks the kDNA. Titration of mitochondrial uncouplers and direct membrane potential measurements reveal that TbTim20 becomes more critical as the membrane potential decreases across all tested cell lines. Proteomic analysis of the uninduced and induced {gamma}L262P TbTim20-RNAi cell line, which lacks kDNA and exhibits the lowest membrane potential, shows depletion of a subset of imported proteins. This subset includes ATPase subunits, suggesting a mechanism by which TbTim20-silenced cell lines become more sensitive to uncouplers. Thus, we propose that TbTim20 supports import of a subset of proteins whose import is hypersensitive to a low membrane potential.

biochemistry↗

bioRxiv2024

Molecular characterization of the permanent outer-inner membrane contact site of the mitochondrial genome segregation complex in trypanosomes

The parasitic protozoan Trypanosoma brucei has a single unit mitochondrial genome linked to the basal body of the flagellum via the tripartite attachment complex (TAC). The TAC is crucial for mitochondrial genome segregation during cytokinesis. At the core of the TAC, the outer membrane protein TAC60 binds to the inner membrane protein p166, forming a permanent contact site between the two membranes. Although contact sites between mitochondrial membranes are common and serve various functions, their molecular architecture remains largely unknown. This study elucidates the interaction interface of the TAC60-p166 contact site. Using in silico, in vitro, and mutational in vivo analyses, we identified minimal binding segments between TAC60 and p166. The p166 binding site in TAC60 consists of a short kinked -helix that interacts with the C-terminal - helix of p166. Despite the presence of conserved charged residues in either protein, electrostatic interactions are not necessary for contact site formation. Instead, the TAC60-p166 interaction is driven by the hydrophobic effect, as converting conserved hydrophobic residues in either protein to hydrophilic amino acids disrupts the contact site. Author summaryMitochondria are surrounded by two membranes and essential for nearly all eukaryotes. Contact sites between the two membranes are important for mitochondrial function. However, most contact sites are dynamic making their molecular architecture challenging to study. The tripartite attachment complex (TAC) of parasitic protozoan Trypanosoma brucei connects its compact mitochondrial genome with the basal body of the flagellum. This couples the segregation of the replicated mitochondrial genome to the old and new basal body. The TAC contains permanent contact sites formed by outer membrane protein TAC60 and the intermembrane space-exposed C-terminus of p166 of the inner membrane. We have used it as a model for a prototypical contact site. AlphaFold predictions and in vitro binding assays identified a small region in the intermembrane space region of TAC60 that binds p166 forming contact sites. In vivo expression of various TAC60 and/or p166 mutants followed by immunoprecipitations demonstrates that contact site formation is driven by the hydrophobic effect and independent of the conserved charged amino acids present at the TAC60-p166 interface. The TAC is unique to Kinetoplastids, understanding the molecular architecture of the TAC60-p166 contact site could therefore inform the development of drugs that disrupt this critical interaction.

biochemistry↗

bioRxiv2024

Intermembrane space-localized twin Cx9C motif-containing TbTim15 is an essential subunit of the single mitochondrial inner membrane protein translocase of trypanosomes

All mitochondria import >95% of their proteins from the cytosol. This process is mediated by protein translocases in the mitochondrial membranes, whose subunits are generally highly conserved. Most eukaryotes have two inner membrane protein translocases (TIMs) that are specialized to import either presequence-containing or mitochondrial carrier pro-teins. In contrast, the parasitic protozoan Trypanosoma brucei has a single TIM complex consisting of one conserved and five unique subunits. Here, we show that the trypanoso-mal TIM complex contains an additional trypanosomatid-specific subunit, designated TbTim15. TbTim15 is associated with the TIM complex, lacks transmembrane domains and localizes to the intermembrane space. TbTim15 is essential for procyclic and bloodstream forms of trypanosomes. It contains two twin CX9C motifs and mediates import of both, presequence-containing and mitochondrial carrier proteins. While the precise function of TbTim15 in mitochondrial protein import is unknown, our results are consistent with the notion that it may function as an import receptor for the non-canonical trypanosomal TIM complex.

biochemistry↗

bioRxiv2022

The mitochondrial genome segregation system of T. brucei: Single p197 molecules connect the basal body with the outer membrane

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body of the flagellum. Here we studied the architecture of the exclusion zone filament of the TAC that connects the basal body with the mitochondrial outer membrane. The only known component of the exclusion zone filaments is p197. Using genetical, biochemical and microscopical methods we show that p197 has three domains all of which are essential for mitochondrial DNA inheritance. The C-terminus of p197 interacts with the mature and pro-basal body whereas its N-terminus binds to the peripheral outer membrane protein TAC65. The large central region of p197 has a high -helical content and likely acts as a flexible spacer. Replacement of endogenous p197 with a functional version containing N- and C-terminal epitope tags together with expansion microscopy demonstrates that p197 alone can bridge the approximately 170 nm gap between the basal body and the periphery of the outer membrane. This demonstrates the power of expansion microscopy which allows to localize distinct regions within the same molecule and suggests that p197 is the TAC subunit most proximal to the basal body. Significance statementSegregation of the replicated single unit mitochondrial genome of Trypanosoma brucei requires a large hardwired structure that connects the organellar DNA with the flagellar basal body. The cytosolic part of this structure consists of filaments made of single p197 molecules, a protein larger than 600 kDa. p197 has three domains all of which are essential for its function. The N-terminus of p197 is anchored to the peripheral outer membrane protein TAC65 whereas its C-terminus connects to the base of the basal body. The large central domain forms an -helix and consists of at least 26 repeats of 175 aa in length. It provides a flexible linker bridging the approximately 170 nm between the outer membrane and the basal body

cell biology↗