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. 2011 May 4;100(9):2184-92.
doi: 10.1016/j.bpj.2011.03.031.

Cardiolipin Affects the Supramolecular Organization of ATP Synthase in Mitochondria

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Free PMC article

Cardiolipin Affects the Supramolecular Organization of ATP Synthase in Mitochondria

Devrim Acehan et al. Biophys J. .
Free PMC article

Abstract

F(1)F(0) ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich in ATP synthase. Our study included a wild-type control, a cardiolipin synthase mutant with nearly complete loss of cardiolipin, and a tafazzin mutant with reduced cardiolipin levels. In the wild-type, the high-curvature edge of crista vesicles was densely populated with ATP synthase molecules that were typically organized in one or two rows of dimers. In both mutants, the density of ATP synthase was reduced at the high-curvature zone despite unchanged expression levels. Compared to the wild-type, dimer rows were less extended in the mutants and there was more scatter in the orientation of dimers. These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the crista membrane.

Figures

Figure 1
Figure 1
State of oligomerization of ATP synthase. (A) 3-D model of a tomogram of a membrane vesicle. F1 particles (n = 187) are shown in random colors and the membrane surface is shown in gray. F1 particles are concentrated at the high-curvature zone of the disc-shaped vesicle. (B) Distance histogram of F1-F1 pairs. The histogram includes all F1-F1 pairs for which the connecting line does not intersect with another F1 particle (see Methods section for details). The distance population peaks at 15 nm and at 23 nm (1 pixel = 8.83 Å). (C) Separate distance histograms are shown for the closest neighbor (blue), the second closest neighbor (green), the third closest neighbor (yellow), the fourth closest neighbor (red), and the fifth closest neighbor (purple). The histogram data points are curve-fitted to enhance clarity. (D) 3-D model of Fig. 1A rendered in different colors. Dimers are colored in alternating shades of red or blue, revealing two parallel dimer rows on opposite sides of the edge of the disc-shaped vesicle. The surface of the vesicle is colored gray. F1 particles on the flat surface of the vesicle are colored white and F1 particles that could not be assigned to any dimer are colored yellow.
Figure 2
Figure 2
Histograms showing (top to bottom) the number of neighbors per F1 particle for wild-type flies (WT), cardiolipin synthase mutant flies (ΔCLS), and tafazzin mutant flies (ΔTAZ). A particle was considered the neighbor of another particle if their distance was within 27 nm and if there was no other particle between the two.
Figure 3
Figure 3
Lipid analysis in ΔCLS Drosophila. (A) Lipids were extracted from wild-type flies (WT) and from cardiolipin synthase mutant flies (ΔCLS) and then underwent HPLC separation. The fractions containing acidic phospholipids were collected and analyzed by MALDI-TOF mass spectrometry in negative ion mode. Mutation of CLS causes a decrease in cardiolipin and an increase in phosphatidylglycerol. The principle peaks include (left to right) palmitoyl-palmitoleoyl-PG (m/z = 719.5), palmitoleoyl-oleoyl-PG (m/z = 745.5), oleoyl-linoleoyl-PG (m/z = 771.5), dipalmitoleoyl-PI (m/z = 805.5), palmitoleoyl-oleoyl-PI (m/z = 833.5), oleoyl-linoleoyl-PI (m/z = 859.5), (C16)3-C18-CL cluster (peak at m/z = 1372), (C16)2-(C18)2-CL cluster (peak at m/z = 1398), C16-(C18)3-CL cluster (peak at m/z = 1422), (C18)4-CL cluster (peak at m/z = 1448). (B) Mitochondria were isolated from wild-type flies (WT) and from cardiolipin synthase mutant flies (ΔCLS). Lipids were extracted from an aliquot corresponding to 2 mg mitochondrial protein and separated by 2-D thin-layer chromatography on silica gel 60 plates developed by chloroform-methanol-20% ammonia (65-30-5) in the first dimension (vertical) and chloroform-acetone-methanol-acetic acid-water (50-20-10-10-5) in the second dimension (horizontal). Lipids were stained with iodine vapor and phospholipids were determined by colorimetric assay of phosphorus after ashing and expressed as percent of total phospholipid. Mutation of cardiolipin synthase decreases cardiolipin in favor of phosphatidylglycerol. CL, cardiolipin; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; Tr, trace; ND, not detected.
Figure 4
Figure 4
Mutation of cardiolipin synthase affects Drosophila physiology at all developmental stages. Physiologic variables were determined in the wild-type strain (WT) and in the mutant strain (ΔCLS). (A) Locomotor activities of larvae were measured by the number of squares (0.25 × 0.25 in2) crossed within 5 min (N = 9 in each group). (B) Pupal heart rates were determined under a light microscope (N = 8 in each group). (C) Longevities of adult flies were compared in a Kaplan-Meier graph (N = 50 in each group). (D) Climbing activities of adult flies (10 days old) were determined by the distance flies climbed within 12 seconds. The graphs show distributions of fly populations over the distance (expressed in 0.5-in units) they climbed within 12 s (triplicate measurements, each performed with 40 flies). (E) Respiratory activities of Drosophila mitochondria (WT, wild-type; ΔCLS, cardiolipin synthase mutant; ΔTAZ, tafazzin mutant) were measured with the BD Oxygen Biosensor System (BD Biosciences, Franklin Lakes, NJ) according to the specifications of the manufacturer. Mitochondria were incubated at a concentration of 1 mg protein/ml in a medium containing 250 mM sucrose, 15 mM KCl, 1 mM EGTA, 5 mM MgCl2, 30 mM KH2PO4, 7 mM succinate, and 2 mM ADP, pH 7.4. Data are the means of two independent experiments. Cardiolipin deficiency is associated with low respiratory activity in ΔTAZ and ΔCLS. F, female; M, male.
Figure 5
Figure 5
Disarray in the supramolecular assembly of ATP synthase in Drosophila mutants with cardiolipin deficiency. (Upper) 3-D models of membrane vesicles from flight-muscle mitochondria of wild-type flies (WT), cardiolipin synthase mutant flies (ΔCLS), and tafazzin mutant flies (ΔTAZ). The lower panel shows corresponding 3-D models, in which neighbors are connected by lines. A particle is considered the neighbor of another particle if their distance from each other is within 27 nm and if there is no other particle in between the two. The models show less connectivity in the supramolecular structure of ATP synthase in the cardiolipin mutants.
Figure 6
Figure 6
2D-BN/SDS-PAGE analysis of ATP synthase in Drosophila mitochondria including wild-type (WT), cardiolipin synthase mutant (ΔCLS), and tafazzin mutant (ΔTAZ). (A) BN-PAGE molecular weight markers. The molecular mass of markers is given in kDa. (B) 2D-BN/SDS-PAGE analysis of the α-subunit of ATP synthase using fluorescing secondary antibody from Li-Cor. In the WT, most of ATP synthase was recovered as dimer (D); in ΔTAZ about equal proportions of ATP synthase were recovered as dimer and as monomer (M); and in ΔCLS most of ATP synthase was recovered as monomer. (C) 2D-BN/SDS-PAGE analysis of the α-subunit of ATP synthase using horseradish peroxidase-conjugated secondary antibody. Some dimers remain present in the ΔCLS mutant.
Figure 7
Figure 7
Structural order in ATP synthase oligomers. (A) Cartoon depicting the orientation of ATP synthase dimers in the membrane plane. The three scenarios include random distribution of isolated dimers (left), nonideal dimer row (middle), and ideal dimer row (right). θ is the angle by which the dimer orientation deviates from the mean direction. (B) Tomographic models of dimer rows in wild-type (WT) and cardiolipin synthase mutant (ΔCLS). (C) Probability density functions. Dimer orientations were measured in tomographic models from WT (n = 75) and ΔCLS (n = 34), yielding a set of angles (θ1, θ2,…, θn). The sets were used to estimate the concentration parameters using Eq. 2. The concentration parameters were κ = 6.1 for WT and κ = 2.4 for ΔCLS. The, probability density functions were calculated from the concentration parameters using Eq. 1.

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