A ribbon diagram of the A k 4 model in complex with A M P and A T P shows conserved regions within the N M P-binding domain highlighted in green, the L I D domain in red, and the P-loop in yellow. The mutated residues K 18, G 89, R 122, N 137, A 166, and T 199 are labeled. The bar graph in panel B displays relative A T P levels in lentiviral-transduced W T and A k 4 K O Listeria-infected B M D M s, normalized to A k 4 W T-transduced A k 4 K O B M D M s. The Western blot below shows A k 4 W T slash mutant and beta-actin protein expression; A K 4 lacks bands corresponding to control and empty vector. Panel C features a bar graph quantifying m t D N A copy number in transduced W T and A k 4 KO T G-p M a c s using q P C R. The graph shows a fluctuating pattern. Panels D and E present bar graphs of the mean fluorescence intensity (M F I) of MitoTracker Green and MitoTracker DeepRed in transduced T G-p M a c s. Both graphs show a fluctuating pattern. Panel F shows a line graph of the oxygen consumption rate (O C R) over time. The graph shows a fluctuating pattern.
Ak4 kinase activity is required for the regulation of mtDNA synthesis. Mock, Ak4 WT, or kinase-dead Ak4 mutants were transduced into Ak4 KO BMDMs or TG-pMacs using lentiviral vectors. Mock-transduced WT macrophages served as controls. Cells were infected with Listeria at an MOI of 5 for 1 h, treated with 250 μg/ml gentamicin, washed with PBS, and maintained in 50 μg/ml gentamicin for either 24 h (C) or 6 h (D–F). (A) A ribbon diagram of the Ak4 model in complex with AMP and ATP is shown. Conserved regions within the NMP-binding domain are highlighted in green, the LID domain in red, and the P-loop in yellow. The mutated residues K18, G89, R122, N137, A166, and T199 are labeled accordingly. (B) Intracellular ATP levels were measured in lentiviral-transduced WT (mock) and Ak4 KO (mock, Ak4 WT, or kinase-dead mutant) Listeria-infected BMDMs (n = 6). ATP levels were normalized to those in Ak4 WT-transduced Ak4 KO BMDMs. Ak4 protein expression was assessed by western blotting (lower panel). (C) mtDNA copy number in transduced WT and Ak4 KO TG-pMacs was quantified by qPCR (n = 3). (D and E) MFI of MitoTracker Green (D) and MitoTracker Deep Red (E) in transduced TG-pMacs was measured by flow cytometry (n = 4). (F) OCR was assessed in transduced cells using an XF-96 analyzer (n = 3). Protein expression levels were normalized to β-actin. mtDNA copy number was normalized to nDNA. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of three independent experiments, and each point represents data from one mouse with two technical repeats. nDNA, nuclear DNA; MFI, mean fluorescence intensity. Source data are available for this figure: SourceData F4.