Mitochondrial nucleoid trafficking regulated by the inner-membrane AAA-ATPase ATAD3A modulates respiratory complex formation

Abstract

Mitochondria have their own DNA (mtDNA), which encodes essential respiratory subunits. Under live imaging, mitochondrial nucleoids, composed of several copies of mtDNA and DNA-binding proteins, such as mitochondrial transcription factor A (TFAM), actively move inside mitochondria and change the morphology, in concert with mitochondrial membrane fission. Here we found the mitochondrial inner membrane-anchored AAA-ATPase protein ATAD3A mediates the nucleoid dynamics. Its ATPase domain exposed to the matrix binds directly to TFAM and mediates nucleoid trafficking along mitochondria by ATP hydrolysis. Nucleoid trafficking also required ATAD3A oligomerization via an interaction between the coiled-coil domains in intermembrane space. In ATAD3A deficiency, impaired nucleoid trafficking repressed the clustered and enlarged nucleoids observed in mitochondrial fission-deficient cells resulted in dispersed distribution of small nucleoids observed throughout the mitochondrial network, and this enhanced respiratory complex formation. Thus, mitochondrial fission and nucleoid trafficking cooperatively determine the size, number, and distribution of nucleoids in mitochondrial network, which should modulate respiratory complex formation.

Keywords: ATAD3A; Drp1; mitochondrial fission; mtDNA nucleoid; respiratory complex.

Fig. 1.

AAA-ATPase domain of ATAD3A interacts with nucleoids in the mitochondrial matrix. (A) Schematic representation of ATAD3A, nucleoids, and mitochondrial fission factors. (B–D) Binding of ATAD3A and TFAM, Mff, or Drp1 in HeLa cells that were transfected with the indicated plasmids. The cells were treated with 1% formaldehyde for 10 min and immunoprecipitation was performed using the cell lysates. The precipitates were analyzed by immunoblotting. (E) HeLa cells were treated with siRNA for Drp1 or control for 96 h. Confocal images of nucleoids and endogenous ATAD3A by immunofluorescence staining of each cell. Insets show a magnified image of each panel. Red, ATAD3A; green, mtDNA. (Scale bars: 10 μm in large panels and 2 μm in Insets.) (F) Confocal images of HA and Tom20 by immunofluorescence staining of HeLa cells transiently expressing C-terminally tagged ATAD3A under conditions in which only the OM was permeabilized (0.04% digitonin) or the OM and IM were both permeabilized (0.2% digitonin). (Scale bar, 10 μm.) (G) Confocal images of the C terminus of ATAD3A, N terminus of ATAD3A, and Tom22 were visualized by immunofluorescence staining in the presence of 0.04% or 0.2% digitonin. (Scale bars, 10 μm.) (H and I) Schematic representation of ATAD3A (H) and TFAM (I) constructs. (J and K) The purified GST-fused proteins and the purified mCherry-fused proteins were separated by SDS/PAGE and stained with Coomassie Brilliant blue (CBB). (L and M) Interaction between ATAD3A and TFAM constructs measured by an on-beads Halo assay. See SI Appendix, Fig. S1C. The signals of mCherry-fused TFAM on GST-ATAD3A (245–586) beads in the presence of ATP or ADP were quantified (M). Steel-Dwass test was performed using Statcel4 software. **P < 0.01. Data are presented as mean ± SD.

Fig. 2.

ATAD3A has a role in mitochondrial nucleoid trafficking, and is critical for the regulation of the size and number of mtDNA nucleoids. (A and B) Confocal images of nucleoids and mitochondria by immunofluorescence staining. Insets show a magnified image of each panel. Red, cytochrome c; green, mtDNA. The proportion of cells containing nucleoid clustering was measured by mtDNA staining (n > 100). (Scale bar: 10 μm in large panels and 2 μm in Insets.) (C) HeLa cells were treated with Drp1 siRNA and/or ATAD3 siRNA for 96 h. Protein levels were determined by immunoblotting using the indicated antibodies. (D) mtDNA content was determined by quantitative PCR. The relative amount of mtDNA (317–381, 65 bp) per nuclear gene (β2M, 95 bp) is shown. (E–H) HeLa cells stably expressing mitochondria-targeted DsRed (mitRFP:red) were treated with the indicated siRNA and stained with SYBR Green I (green) for 5 min and live-cell images were obtained by spinning-disk confocal fluorescence microscopy. Images were taken every 5 s for 10 min. Arrowheads in E–H indicate randomly selected and tracked nucleoids in a part of live cell imaging shown in Movies S1–S4. The color of each arrowhead corresponds to the color of each trajectory in I. (Scale bar, 1 μm.) (I) Trajectories of nucleoids in E–H are shown. The color of each trajectory corresponds to the arrowhead of each color in E–H. (Scale bar, 1 μm.) Also see Movie S5. (J) The moving distance migrated in 10 min of each randomly selected nucleoid is shown. Tukey–Kramer multiple comparisons test was performed using Statcel4 software. *P < 0.05, **P < 0.01. Data are represented as mean ± SD in all graphs.

Fig. 3.

ATPase domain and coiled-coil domains for ATAD3A oligomerization are required for nucleoid trafficking. (A) Schematic representation of ATAD3A mutants. (B) Binding between WT or coiled-coil mutant (LPAP) ATAD3A in HeLa cells transfected with the indicated plasmids. The cells were harvested and immunoprecipitation was performed using the cell lysates. The precipitates were analyzed by immunoblotting. (C) Rescue experiments on nucleoid enlargement and clustering. HeLa cells stably expressing mitRFP and WT or mutant ATAD3A were treated with the indicated siRNA or control for 96 h. Confocal images of nucleoids and mitochondria by immunofluorescence staining are shown. Red, mitRFP; green, mtDNA. (Scale bar, 2 μm.) (D) Quantification of cells with clustered nucleoids in C. Tukey–Kramer multiple comparisons test was performed using Statcel4 software. **P < 0.01 (vs. Drp1 RNAi). Data are represented as mean ± SD. (E) Rescue experiments of nucleoid dynamics. HeLa cells stably expressing mitRFP and WT or mutant ATAD3A were treated with siRNA for ATAD3 or control for 96 h. The cells were stained with SYBR Green I for 5 min and live-cell images were obtained by spinning-disk confocal fluorescence microscopy. The images were obtained every 5 s for 10 min. Red, mitRFP; green, mtDNA. (Right) Trajectories of randomly selected nucleoids. Also see Movie S6. (Scale bar, 5 μm.) (F) The moving distance migrated in 10 min for each randomly selected nucleoid is shown. Tukey–Kramer multiple comparisons test was performed using Statcel4 software, **P < 0.01 (vs. cont RNAi). Box-and-whisker plots were represented minimum to maximum. (G) Schematic representation of nucleoid trafficking is shown. Nucleoid movement requires an active ATPase domain in the mitochondrial matrix and coiled-coil domains in the IMS of ATAD3A.

Fig. 4.

Nucleoid distribution affects the stabilization of respiratory subunits. (A) Drp1 KO HeLa cells were treated with siRNA for ATAD3 or control for 96 h. The cells were stained with MitoTracker Red for 15 min and SYBR Green I for 5 min. The cells were observed by fluorescence microscopy. (Scale bar, 20 μm in large panels and 10 μm in Insets.) (B) Protein levels of respiratory subunits were determined by immunoblotting using the indicated antibodies. Drp1 KO HeLa cells were treated with siRNA for ATAD3 or control for 144 h. Cells treated with 10 μM 2′,3′-Dideoxycytidine (ddC) for 168 h were used as controls. (C) The graph shows the relative protein level of each OXPHOS component at ATAD3 RNAi for 96 h against control. Multiple comparison with Tukey’s test was performed using GraphPad Prism version 6.00. *P < 0.05, **P < 0.01, ***P < 0.001 (vs. each subunit of cont RNAi). Data are represented as mean ± SD. (D) HeLa cells treated as described in C were determined by immunoblotting using the indicated antibodies. (E) The relative protein level of each OXPHOS component is shown graph as described in C. Unpaired t test with Welch’s collection was performed using GraphPad Prism v6.00. *P < 0.05, **P < 0.01 (vs. each subunit of cont RNAi). (F) Schematic representation of the mechanism and the role of mtDNA nucleoid distribution regulated by ATAD3A and Drp1. The accumulation of clustered nucleoids in mitochondrial fission-deficient cells formed by active nucleoid trafficking mediated by ATAD3A leads to the reduced respiratory complexes.