Fig 1.
γATPase editing yielded known and novel oligomycin-resistantmutants.
(A) The schematic illustrates oligo targeting for saturation mutagenesis of the T. b. brucei γATPase M282 residue. A sixty-four fold degenerate ssODN was transfected into wild-type T. b. brucei cells, followed by oligomycin selection and γATPase amplicon-sequencing. (B) The heat map shows relative representation of each possible amino acid variant at the targeted M282 site and at adjacent sites; averages for two independent oligomycin-resistant cultures relative to an unedited control. More than 8 M reads were mapped per site on average. Unedited codons are indicated (black) as is the previously reported M282L mutation (red outline). (C) The Sanger sequencing traces show single allele edits encoding the L262P, A273P and M282F mutations, each involving a double nucleotide edit; edited nucleotides are indicated by asterisks. (D) Simplified schematic of the trypanosome F1FO-ATP synthase and key components discussed here. Names of F1 subunits are in white letters (F1 subunits p18, delta and epsilon were omitted for simplicity). The α/β hexamer (cyan) is held in place by attachment to the peripheral ‘stator’ stalk (green) via the OSCP subunit (orange). The proton-translocating part consists of the c10-ring (grey) and the kDNA-encoded A6 subunit (red). PMS, peripheral membrane subcomplex (blue); IMM, inner mitochondrial membrane. The AlphaFold models for wild-type (WT) and mutant γATPase were generated using the AlphaFold server, showing mutant residues in green.
Fig 2.
Only bi-allelic γATPase M282 editing yielded acriflavine-resistant mutants.
(A) The Sanger sequencing traces show T. b. brucei γATPase M282F edits, both heterozygous and homozygous. A GCT/G, alanine polymorphism can be seen on the right-hand side of each panel, confirming retention of both alleles. Edited nucleotides are indicated by asterisks. (B) Dose-response curves for oligomycin, measured in duplicate. EC50 values are shown. (C) Dose-response curves for acriflavine, measured in duplicate. EC50 values are shown.
Fig 3.
ATP synthase remodelling in homozygousγATPase mutants.
(A) Proteomics analysis of wild-type T. b. brucei and homozygous γATPase M282F mutants. Subunits of the F1 and Fo γATPase components are highlighted. Averages from three replicates; n = 6847 proteins. (B) The microscopy images show nuclei (larger structures) and kDNA (smaller structures) in wild-type T. b. brucei and homozygous γATPase M282F mutant cells; DNA was stained with DAPI. Scale bars, 5 μm. (C) Flow cytometry analysis of MitoTracker-stained cells, wild-type and homozygous γATPase M282F mutants. The data are representative of three technical replicates. Control, unstained cells.
Fig 4.
kDNA loss in homozygous γATPasemutants.
(A) The microscopy images show homozygous γATPase M282F mutant cells with or without kDNA, the smaller blue DNA-stained structures. DNA was stained with DAPI. Scale bars, 5 μm. (B-C) Whole genome sequencing data for wild-type (WT) T. b. brucei, the homozygous γATPase M282F mutant with kDNA (P for parent) and independently generated clones lacking kDNA. (B) The upper circular plot shows genome sequencing data mapped to the T. b. brucei nuclear chromosomes 1–11 (grey). The lower circular plot shows genome sequencing data mapped to the T. b. brucei kDNA (magenta), maxicircle sequence and the most abundant minicircle sequences; the numbers indicate minicircle ID. Mapping is for 150-bp bins and for two independently generated kDNA negative clones. (C) The heatmap shows data for maxicircle sequence, additional minicircle sequences (n = 90), and for all four kDNA negative clones. kDNA negative clone numbers are indicated in each panel.
Fig 5.
Proteomic and mitochondrial impacts of kDNA loss.
(A) Proteomics analysis of homozygous γATPase M282F mutants with and without kDNA with subunits of the F1 and Fo γATPase components highlighted. Averages from three replicates; n = 6768 proteins; clone 1.2 is shown here and three other clones are shown in Supplementary Fig 2. (B) The representative super resolution microscopy images show wild-type T. b. brucei and homozygous γATPase M282F mutant cells lacking kDNA, following growth in acriflavine. DNA was stained with DAPI (cyan) and mitochondria were stained with MitoTracker (yellow). Nuclear DNA (N) and kDNA (K) are indicated. Scale bars, 2 μm. A gallery of additional images is shown in Supplementary Fig 3. (C) Flow cytometry analysis of MitoTracker-stained cells, homozygous γATPase M282F mutants with or without kDNA. Data are shown for two independent biological replicates without kDNA and are representative of three technical replicates in each case.
Fig 6.
Mitochondrial proteome remodelling following kDNA loss.
(A) Proteomics analysis of homozygous γATPase M282F T. b. brucei mutants and all four M282F clones lacking kDNA. The boxplot shows nuclear proteins (n = 1185, GO:0005634) and mitochondrial proteins (n = 1431, GO:0005739). Boxes indicate the interquartile range (IQR) and the whiskers show the range of values within 1.5 × IQR. (B) Proteomics analysis showing all proteins with log2 average expression >16 and with some notable proteins highlighted. (C) Gene Ontology profiles for proteins with log2 average expression >16 that are significantly (>2 –log10 FDR) decreased or increased in abundance in kDNA negative cells. Mito’, mitochondrial. (D) Selected cohorts of proteins that are significantly decreased or increased in abundance in kDNA negative cells. Boxes indicate the interquartile range (IQR) and the whiskers show the range of values within 1.5 × IQR. MCP, mitochondrial carrier proteins; TIM, Translocases of the Inner Membrane; ATOM, Archaic Translocase of the Outer Membrane; MICOS, Mitochondrial contact site and Cristae Organizing System; POMP, Present in the Outer mitochondrial Membrane Proteome; EMC, ER-Membrane Complex; ER, Endoplasmic Reticulum. Cohorts were derived using GO-terms except for kinetoplast-associated proteins from [31] and MCP, TIM and POMP, derived using wild-card searches, MCP*, TIM* and POMP* at https://tritrypdb.org [43]. Data are shown for kDNA negative clones 1.2 (blue) and 2.1 (red).