A comparison of three approaches for the discovery of novel tripartite attachment complex proteins inTrypanosoma brucei

Author summary Trypanosoma bruceibelongs to a group of organisms that exist as human, animal and plant parasites.T.brucei(a human and animal parasite) has been developed as a model system to study basic biological as well as disease related questions in this group of organisms. We study how the parasite duplicates and divides its mitochondrial genome, an essential component of its energy generating machinery. The structure involved in dividing the mitochondrial genome into the daughter cells during cell division is called the tripartite attachment complex (TAC). The TAC is likely a unique structure not present in the host and thus might provide a new avenue for drug development. In this manuscript, we compare different techniques that allow the identification of novel components of this structure and verify the localisation of some of them. Furthermore, we also establish the interaction of two previously identified protein components. Trypanosoma bruceiis a single celled eukaryotic parasite and the causative agent of human African trypanosomiasis and nagana in cattle. Aside from its medical relevance,T.bruceihas also been key to the discovery of several general biological principles including GPI-anchoring, RNA-editing and trans-splicing. The parasite contains a single mitochondrion with a singular genome. Recent studies have identified several molecular components of the mitochondrial genome segregation machinery (tripartite attachment complex, TAC), which connects the basal body of the flagellum to the mitochondrial DNA ofT.brucei. The TAC component in closest proximity to the mitochondrial DNA is TAC102. Here we apply and compare three different approaches (proximity labelling, immunoprecipitation and yeast two-hybrid) to identify novel interactors of TAC102 and subsequently verify their localisation. Furthermore, we establish the direct interaction of TAC102 and p166 in the unilateral filaments of the TAC.