Conserved cardiolipin-mitochondrial ADP/ATP carrier interactions assume distinct structural and functional roles that are clinically relevant

Figures

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  Fig. 1:. Generation and characterization of yeast Aac2 CL-binding mutants. (A) Strategy to disrupt CL-binding sites in Aac2. Aac2 was modeled onto bovine ANT1 (PDB ID: 2C3E) using SWISS-MODEL. Negatively charged amino acids introduced (light orange) into Aac2 CL-binding motifs face toward the CL phosphate headgroup (orange). (B) Expression of WT and mutant Aac2 was detected in whole cell extracts by immunoblot; Kgd1 and Tom70 served as loading controls (n=5). Significant difference was obtained by one-way ANOVA with Dunnett’s multiple comparisons test (vs. WT) ***p<0.001. (C) Growth phenotype of Aac2 CL-binding mutants. Serial dilutions of indicated cells were spotted onto fermentable (YPD) and respiratory (YPEG) media and incubated at 30 or 37°C for 3–5 days (n=3). (D) Membrane topology of WT and mutant Aac2. Isolated mitochondria were osmotically ruptured and treated with or without proteinase K as indicated. Aac2 N-terminus was detected by a monoclonal antibody 6H8 recognizing the first 13 amino acids MSSNAQVKTPLPP (n=6). IMS, intermembrane space; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. In B-D, representative images from the indicated replicates are shown.

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  Fig. 2:. Native mass spectrometry analysis to detect lipid-protein interaction of Aac2. (A) FlagAac2 affinity purified from WT mitochondria was associated with CATR and up to three CL molecules. (B) FlagAac2 affinity purified from crd1Δ mitochondria which lack CL did not co-purify other phospholipids. Insets in A and B show the fractional population of FlagAac2 relative to FlagAac2+CATR when immunoprecipitated from WT and crd1Δ mitochondria, respectively. Mean with SEM (n=3). Significant differences as determined by Student’s t-test indicated (****p<0.0001). (C) CL, PE, PG, or lyso-PC was added to FlagAac2 purified from crd1Δ mitochondria at the indicated concentrations. (D) Fractional population of FlagAac2, FlagAac2+CATR alone or associated with one to three CL molecules was determined for WT and Aac2 CL-binding mutants. Mean with SEM (n=5–6). Significant differences were obtained by one-way ANOVA with Dunnett’s multiple comparisons test (* vs. FlagAac2; † vs. FlagAac2+CATR+1CL); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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  Fig. 3:. CLs associated with Aac2 stabilize its monomeric structure. Mitochondria (100 μg) from WT and Aac2 CL-binding mutants were mock-treated or instead incubated with either 40 μM CATR (A) or 10 μM BKA (B) and then solubilized with 1.5% (w/v) digitonin. The extracts were resolved by 6 to 16% blue native-PAGE and immunoblotted for Aac2 (n=6). Representative images from the indicated replicates are shown.

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  Fig. 4:. Distinct functional roles of the three tightly bound CLs. (A-C) ADP/ATP exchange: The efflux of matrix ATP was detected with isolated mitochondria as NADPH formation (A340; absorbance at 340 nm) occurring coupled with an in vitro glycolysis reaction which contained glucose, hexokinase, and glucose-6-phosphate dehydrogenase. The reaction was initiated by adding ADP. The measurement was performed in the presence of 5 mM malate and 5 mM pyruvate (+Mal/Pyr). Where indicated, WT mitochondria were treated with 5 μM CATR prior to the efflux reaction. (A) The linear part of the initial velocity for the ATP efflux was plotted and curve fitting performed by nonlinear regression (mean with SEM, n=6). Plots of aac2Δ and WT are repeated in all panels. (B) The initial velocity following the addition of 33 μM ADP presented as scatter plots (mean with SEM, n=6). (C) Fitted Km and Vmax values were obtained using the Michaelis-Menten equation (mean). The range of determined values is shown in brackets (n.d., not detected). (D) Oxygen consumption rate (OCR) in isolated mitochondria with sequential injections of respiratory substrate (NADH or succinate), ADP, oligomycin, and CCCP was measured using…

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  Fig. 5. Disturbed Aac2-CL interaction is a pathological mechanism. (A) Mitochondrial myopathy patient mutation L155F was introduced into yeast Aac2 as Fig. 1A. (B) Native MS analysis obtained the fractional population of WT and indicated FlagAac2 mutants associated with CATR and one to three CL molecules. (C) Expression of WT and mutant Aac2 was detected in whole cell extract by immunoblot. Kgd1 and Tom70 served as loading controls (n=4). (D) Growth phenotype of Aac2 CL-binding mutants. Serial dilutions of indicated cells were spotted onto fermentable (YPD) and respiratory (YPEG) media and incubated at 30 or 37°C for 3–5 days (n=4). (E) Aac2 monomeric structure: 100 μg of mitochondria from WT and Aac2-binding mutants were mock-treated or instead incubated with either 40 μM CATR (C) or 10 μM BKA (B) and then solubilized with 1.5% (w/v) digitonin. The extracts were resolved by 6 to 16% blue native-PAGE and immunoblotted for FlagAac2 (n=6). (F-H) ADP/ATP exchange: The efflux of matrix ATP was detected with isolated mitochondria as NADPH formation (A340; absorbance at 340 nm) occurring coupled with in vitro glycolysis reaction as Fig. 4. The measurement was performed in the presence of 5 mM…

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  Fig. 6. Human ANT1 L141F are structurally and functionally compromised. (A) Human ANT1 was modeled onto bovine ANT1 (PDB ID: 2C3E) using SWISS-MODEL. (B-F) FlagANT1 and the indicated ANT1 mutants were induced by 0.25 μg/ml doxycycline in antnull T-REx-293 cells with three ANT isoforms (ANT1, 2, and 3) knocked out. (B) Expression of ANT1 was detected in whole cell extracts by immunoblot. β-actin and GRP75 were loading controls (n=3). (C) ANT1 monomeric structure: 80 μg of mitochondria from WT and ANT1 mutants were mock-treated or instead incubated with either 40 μM CATR or 10 μM BKA and then solubilized with 1.5% (w/v) digitonin. The extracts were resolved by 6 to 16% blue native-PAGE and immunoblotted for FlagANT1 (n=3). (D-E) ADP/ATP exchange: The efflux of matrix ATP was detected with isolated mitochondria as NADPH formation (A340; absorbance at 340 nm) as Fig. 4. The measurement was performed in the presence of 5 mM malate and 5 mM pyruvate. 5 μM CATR was added to WT mitochondria prior to stimulating the efflux (n=3). (D) The initial velocity following the addition of ADP at indicated concentrations was plotted (mean with SEM). Curve fitting was performed by nonlinear regression.…

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  Fig. 7:. MD simulations predict reduced CL-ANT1 affinity at pocket 2 for L141 mutants. (A-B). Time-averaged 2D density maps of CL lipids for the equilibrium prebound (A) and unbound (B) simulations. The CL binding pockets are defined by the blue (pocket 1), cyan (pocket 2) and green (pocket 3) dots, which represent the amino acids that comprise each binding site. The density surfaces were calculated using vmd volmap with 1 Å resolution. Occupancy scale bar on right applies to all images. (C-D) Radial distribution function profile for the identification of CL lipids around residue 141 for prebound (C) and unbound (D) simulation. (E) The estimated relative free energy required to decouple CL from binding pocket 2 of ANT1 for WT and mutants L141F and L141E. Using Wilcoxson rank sum test between WT and L141E indicates statistical significance (p< 0.05), while the difference between WT and L141F is not significant. Note: Binding site residues considered in the present study are based on the protein-CL lipid interactions and are the selected amino acid residues within 8 Å of the bound CL molecules: Pocket 1: 36, 53, 54, 55, 271, 272, 273, 274, 275, and 276; Pocket 2: 71, 72, 73, 74, 75, 141,…