Molecular characterization of the permanent outer-inner membrane contact site of the mitochondrial genome segregation complex in trypanosomes
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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 α-helix that interacts with the C-terminal α- 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 summary
Mitochondria 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.
