The kinetoplast incorporates the large mitochondrial genome present in the eponymous Kinetoplastida. Trypanosoma brucei is an African trypanosome that can lose kinetoplast DNA (kDNA), however, when the nuclear-encoded gamma subunit of the mitochondrial F 1 F O -ATP synthase (γATPase) is mutated. These mutations, analogous to a broken camshaft at the core of the ATP synthase rotary motor, are associated with multidrug resistance, and correlated with tsetse-fly independent mechanical transmission, and geographical spread of these parasites beyond Africa. Here we engineer kDNA-independent T. brucei to explore origins and consequences of kDNA loss. We use oligo targeting to edit the native γATPase gene, and selection with the ATP synthase targeting drug oligomycin to enrich the desired mutants. Using this approach, we identify novel M 282 F, M 282 W, and M 282 Y mutants, and subsequently generate precision-edited strains expressing the previously described L 262 P or A 273 P mutants, or the novel M 282 F mutant. Heterozygous M 282 F mutants retain sensitivity to the kDNA-targeting drug acriflavine, while homozygous M 282 F mutants are acriflavine resistant. Proteomic analysis of the kDNA-positive homozygous M 282 F mutant reveals highly specific depletion of ATP synthase-associated proteins, but not the F 1 subunits. Proteomic analysis following acriflavine-induced kDNA loss then reveals depletion of kDNA-binding proteins and mitochondrial RNA-processing factors alongside increased expression of mitochondrial membrane-associated transporters. We conclude that T. brucei cells with a homozygous γATPase M 282 F mutation remodel ATP synthase subunit expression and readily tolerate kDNA loss, which is accompanied by substantial remodelling of the mitochondrial proteome.
Ridgway et al. (Wed,) studied this question.