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Cardiolipin, conformation, and respiratory complex-dependent oligomerization of the major mitochondrial ADP/ATP carrier in yeast

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Science Advances  28 Aug 2020:
Vol. 6, no. 35, eabb0780
DOI: 10.1126/sciadv.abb0780
  • Fig. 1 CL stabilizes the tertiary structure and quaternary assembly of Aac2 and controls its conformation.

    (A) Mitochondria from WT or crd1∆ yeast were supplemented with PC or increasing amounts of CL (relative amount added indicated) before solubilizing them with an optimized concentration of digitonin to decrease differences in Aac2 solubilization efficiency: 1.0% (w/v) for mock (−) or CL-treated crd1Δ mitochondria, 1.5% (w/v) for mock (−) or CL-treated WT mitochondria and PC-treated crd1Δ mitochondria, and 2.0% (w/v) for PC-treated WT mitochondria. Mitochondrial extracts were resolved by 6 to 16% blue native polyacrylamide gel electrophoresis (BN-PAGE) and immunoblotted for Aac2. Red arrowheads highlight CL-restored assembly of Aac2 with RSCs. Aac21, Aac2 monomer; III2IV2-Aac2 and III2IV-Aac2, Aac2 associated with large and small RSCs, respectively; 4° and 3°, Aac2 quaternary and tertiary assembly, respectively (n = 3). (B) WT and crd1∆ mitochondria (100 μg), mock-treated (M) or instead incubated with BKA (10 μM) or CATR (40 μM), were solubilized with 1.5% (w/v) digitonin, resolved by 6 to 16% BN-PAGE, and immunoblotted for Aac2 (n = 3). (C) WT and crd1∆ mitochondria (100 μg) were incubated with buffer, CATR (40 μM), or BKA (10 μM) and then solubilized with 1.5% (w/v) digitonin containing increasing amounts of trypsin (0, 0.5, 5, and 50 μg/ml) for 30 min on ice. After trypsin inactivation, the clarified extracts were resolved by 10 to 16% SDS-PAGE and immunoblotted as designated. For Aac2 detection, an Aac2 monoclonal antibody 2C10 was used that detects the following internal epitope (NH2-IVAAEGVGSLFKG-COOH). (n = 3). (D) Model of the predicted trypsin site in Aac2. Aac2 in the c-state [Protein Data Bank (PDB) ID: 4C9G] or modeled in the m-state (based on PDB ID: 6GCI). The left two panels are the two conformational states (as indicated) viewed from the side, and the right two panels are the indicated conformational states viewed from the bottom (matrix facing). The 2C10 epitope is shown in yellow, CL in green, R191 in cyan, and R204 in blue. IMS, intermembrane space. (E) Schematic depicting role of CL on Aac2 conformation.

  • Fig. 2 Aac2 oligomerization is conformation sensitive.

    (A) Schematic representation of yeast strains expressing endogenously tagged Aac2. (B) Fluorescent immunoblots of whole-cell extracts from indicated haploid yeast strains for Aac2 (red) and either the HA or FLAG epitopes (green); Tom70 served as loading control. *, nonspecific bands (n = 3). (C) Serial dilutions of haploid cells from indicated strains were spotted onto YP medium supplemented with sucrose YP-Sucrose or ethanol-glycerol (YPEG) and incubated at 30°C for 3 days (n = 3). (D) Mating strategy to establish diploid yeast expressing two different endogenously tagged forms of Aac2. (E) Diploid mitochondria (20 μg) were resolved by 10 to 16% SDS-PAGE and immunoblotted as indicated. The migration of epitope-tagged and WT Aac2, which were codetected with an Aac2 polyclonal antisera, is indicated. *, nonspecific bands. Bottom panel shows Ponceau S–stained membrane (n = 3). (F) Mitochondria (250 μg) from the indicated CL-producing strains, preincubated with CATR (40 μM) or BKA (10 μM) as listed, were solubilized with 1.5% (w/v) digitonin and FLAG-Aac2 immunoprecipitated (IP) using anti-FLAG resin. The presence of copurified HA-Aac2 and subunits of complexes III (Cor1, Cor2, Rip1, and Qcr6) and IV (Cox1 and Cox4) was determined by immunoblotting; Atp1, Atp2, and Kgd1 served as controls. *, nonspecific bands. Four percent of input (mitochondria) and unbound (flow through following FLAG immunoprecipitation) was analyzed (n = 4). (G) The amount of HA-Aac2 and respiratory complex subunits coimmunoprecipitated with FLAG-Aac2 in untreated mitochondria was determined relative to mitochondria preincubated with CATR (means ± SEM for n = 4 independent experiments). (H) The amount of HA-Aac2 and respiratory complex subunits coimmunoprecipitated with FLAG-Aac2 in BKA pretreated mitochondria was determined relative to mitochondria preincubated with CATR (means ± SEM for n = 8 independent experiments). Statistical differences for (G) and (H) were determined by Mann-Whitney rank sum test.

  • Fig. 3 CL-dependent Aac2 oligomerization cannot be rescued with protein-stabilizing Aac2 inhibitors.

    (A) Yeast strains were grown in YP-Sucrose medium supplemented with 32P (2.5 μCi/ml) overnight. Phospholipids were extracted and separated by thin-layer chromatography (TLC). The migration of PC, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidic acid (PA), and CL is indicated (n = 6). (B) WT or crd1∆ mitochondria (250 μg), preincubated with CATR (40 μM) or BKA (10 μM) as listed, were solubilized with 1.5% (w/v) digitonin and FLAG-Aac2 immunoprecipitated using anti-FLAG resin. The presence of copurified HA-Aac2 and subunits of complexes III (Cor1, Cor2, Rip1, and Qcr6) and IV (Cox1 and Cox4) was determined by immunoblotting; Atp1, Atp2, and Kgd1 served as controls. *, nonspecific bands. Proteins that copurified with FLAG-Aac2 when CL-containing mitochondria were preincubated with CATR (red arrowheads) or BKA (blue arrowheads) are marked. Four percent of input (mitochondria) and unbound (flow through following FLAG immunoprecipitation) was analyzed (n = 4). (C) The amount of HA-Aac2 and respiratory complex subunits coimmunoprecipitated with FLAG-Aac2 in BKA or CATR pretreated CL-null mitochondria was determined relative to similarly treated CL-containing mitochondria (means ± SEM for n = 4 independent experiments). Statistical differences were determined by Mann-Whitney rank sum test.

  • Fig. 4 Aac2 oligomerization is specific.

    (A) WT mitochondria (100 μg), untreated (−) or instead incubated with CATR (40 μM) or BKA (10 μM), were solubilized with digitonin [1.5% (w/v)] or increasing amounts of DDM [0.32 and 0.64% (w/v)], resolved by 6 to 16% BN-PAGE, and immunoblotted for Aac2 (top), complex III (Rip1, middle), or complex IV (Cox4, bottom). Aac21, Aac2 monomer; III2IV2-Aac2 and III2IV-Aac2, Aac2 associated with large and small RSCs, respectively (n = 4). (B) WT mitochondria (250 μg), preincubated with CATR (40 μM) as listed, were solubilized with digitonin [1.5% (w/v)] or increasing amounts of DDM [↓ = 0.32% and ↑ = 0.64% (w/v)], and FLAG-Aac2 immunoprecipitated using anti-FLAG resin. The presence of copurified HA-Aac2 and subunits of complexes III (Cor1, Cor2, Rip1, and Qcr6) and IV (Cox1 and Cox4) was determined by immunoblotting; Atp1, Atp2, Om45, and Kgd1 served as controls. *, nonspecific bands. Four percent of input (mitochondria) and unbound (flow through following FLAG immunoprecipitation) was analyzed (n = 3).

  • Fig. 5 RSCs are required for Aac2 multimerization.

    (A) WT (+mtDNA) and rho- (−mtDNA) mitochondria were solubilized with digitonin, resolved by BN-PAGE, and immunoblotted for complex III, complex IV, or Aac2. Aac21, Aac2 monomer; III2IV2-Aac2 and III2IV-Aac2, Aac2 associated with large and small RSCs, respectively; 4° and 3°, Aac2 quaternary and tertiary assembly, respectively (n = 3). (B) Mitochondrial phospholipids separated by TLC and revealed by phosphorimaging. (C) The relative abundance of CL (means ± SEM for n = 6). Significant differences were determined by Student’s t test (HA/FLAG + mtDNA versus HA/FLAG-mtDNA) or Mann-Whitney rank sum test (HA/WT + mtDNA versus HA/WT-mtDNA). n.s., not significant. (D) Immunoblots of diploid mitochondria. *, nonspecific bands (n = 4). (E) Aac2 level as a function of mtDNA was determined (means ± SEM for n = 4). (F) The indicated diploid mitochondria were preincubated with CATR, solubilized with digitonin, and FLAG-Aac2 immunoprecipitated. Different quantities of mitochondria that nonetheless contained equivalent amounts of Aac2 (WT, HA-Aac2, and/or FLAG-Aac2; aac2∆ mitochondria were used to normalize the total amount of protein included in each sample) were used for these immunoprecipitations. The presence of copurified HA-Aac2 and subunits of complexes III and IV was determined; Atp1, Atp2, and Kgd1 served as controls. *, nonspecific bands. Four percent of input (mitochondria) and unbound (flow through following FLAG immunoprecipitation) was analyzed (n = 4). (G) The amount of HA-Aac2 coimmunoprecipitated with FLAG-Aac2 without mtDNA was determined relative to mtDNA-containing mitochondria (means ± SEM for n = 4 independent experiments). The statistical difference was determined by Mann-Whitney rank sum test.

  • Fig. 6 Multiple copies of Aac2 independently associate with complexes III and IV.

    (A) Mitochondria from the indicated haploid strains were resolved by BN-PAGE (top three panels) to assess complex assembly and SDS-PAGE (bottom three panels) to confirm genotype. Aac2-containing complexes that comigrate with complex III in the absence of assembled complex IV (red arrowheads) or with complex IV in the absence of complex III (blue arrowheads) are highlighted. Aac21, Aac2 monomer (n = 3). (B) Mitochondrial phospholipids separated by TLC and revealed by phosphorimaging. (C) The relative abundance of CL (means ± SEM for n = 6). Significant differences relative to WT were determined by one-way analysis of variance (ANOVA) with Holm-Sidak pairwise comparisons. (D) Immunoblots of diploid mitochondria. *, nonspecific bands (n = 3). (E) The indicated diploid mitochondria were preincubated with CATR, solubilized with digitonin, and FLAG-Aac2 immunoprecipitated. The presence of copurified HA-Aac2 and subunits of complexes III and IV was determined; Atp1, Atp2, Por1, and Kgd1 served as controls. Four percent of input (mitochondria) and unbound (flow through following FLAG immunoprecipitation) was analyzed (n = 3). (F) Cartoons depicting the two structural levels supported by CL for Aac2 assembly. In membranes that contain CL, CL stabilizes the Aac2 tertiary fold and is essential for the interaction of Aac2 with RSCs (note that Aac2 and RSCs are not drawn to scale). In the absence of CL, the tertiary and quaternary assembly of Aac2, as well as its activity, is compromised.

Supplementary Materials

  • Supplementary Materials

    Cardiolipin, conformation, and respiratory complex-dependent oligomerization of the major mitochondrial ADP/ATP carrier in yeast

    N. Senoo, S. Kandasamy, O. B. Ogunbona, M. G. Baile, Y. Lu, S. M. Claypool

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