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

PLoS Negl Trop Dis. 2020 Sep 16;14(9):e0008568. doi: 10.1371/journal.pntd.0008568. eCollection 2020 Sep.

Abstract

Trypanosoma brucei is a single celled eukaryotic parasite and the causative agent of human African trypanosomiasis and nagana in cattle. Aside from its medical relevance, T. brucei has 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 of T. 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Cattle Diseases / parasitology
  • DNA, Kinetoplast / metabolism
  • DNA, Mitochondrial / genetics*
  • Flagella / metabolism
  • Genome, Mitochondrial / genetics
  • Humans
  • Immunoprecipitation*
  • Mitochondria / genetics
  • Mitochondria / physiology
  • Protozoan Proteins / genetics*
  • Trypanosoma brucei brucei / genetics
  • Trypanosomiasis, African / veterinary
  • Two-Hybrid System Techniques*

Substances

  • DNA, Kinetoplast
  • DNA, Mitochondrial
  • Protozoan Proteins

Grant support

TO was supported by SNF179454, Swiss National Science Foundation, http://www.snf.ch. HB was supported by SNF179454, Swiss National Science Foundation, http://www.snf.ch, Canton of Bern. LP was supported by SNF179454, Swiss National Science Foundation, http://www.snf.ch. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.