Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast

Abstract

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.

Keywords: Bioenergetics/Electron Transfer Complex; Cardiolipin; Membrane Biogenesis; Membrane Lipids; Mitochondria; Mitochondrial Diseases; Phospholipid; Yeast.

FIGURE 1.

CLD1 is epistatic to TAZ1 in the CL remodeling pathway by mitochondrial phospholipid analysis. A, mitochondrial phospholipids from the indicated strains derived from GA74-1A were labeled with ³²P_i and separated by TLC. B, quantification of A. n = 5–6. ***, p < 0.001. Mitochondrial phospholipids from the indicated strains derived from C PTY144 and D W303 were analyzed as in A. Whole cell extracts from the indicated strains derived from E PTY144 and F W303 were immunoblotted. * indicates a nonspecific cross-reaction of the Cld1p antisera. PA, phosphatidic acid; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine.

FIGURE 2.

CLD1 is epistatic to TAZ1 in the CL remodeling pathway by respiratory growth analysis. 1:4 serial dilutions of the indicated strains derived from GA74-1A (A), PTY144 (B), or W303 (C) were spotted on the indicated media and incubated at 30 or 37 °C.

FIGURE 3.

Δcld1 contains unremodeled CL. A, acyl chain composition of CL was determined by multidimensional mass spectrometric array analysis. n = 3. B, CL was categorized by the number of saturated acyl chains and expressed as a % of the total CL. C, quantification of the total amount of CL per mg of protein. D, CL was categorized by the number of saturated acyl chains and expressed as the amount of CL per mg of protein. E, acyl chain composition of MLCL was determined by multidimensional mass spectrometric array analysis. n = 3. Statistical analysis is provided in supplemental Table S1.

FIGURE 4.

Mitochondrial morphology is not affected by unremodeled CL. Mitochondria from the indicated strains derived from GA74 (A–E) and W303 (F–J) yeast were analyzed by transmission electron microscope. A and F, representative micrographs from the indicated strains. m, mitochondria; n, the nucleus. Bars, 0.5 μm. B and G, quantification of cristae length per mitochondrion. C and H, quantification of outer membrane length per mitochondrion. D and I, quantification of aberrant mitochondria for each strain, defined as the appearance of exaggerated cristae >0.5 μm in length. The number of mitochondria analyzed is indicated for each strain. E and J, examples of mitochondria with exaggerated cristae. Bars, 0.5 μm. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 5.

OXPHOS function is not affected by unremodeled CL. A, mitochondria were solubilized with digitonin, separated by blue native-PAGE (top panels), and immunoblotted for Cyt1p (complex III), Cox2p (complex IV), Atp2p (complex V), and Aac2p. Bottom panels are immunoblots following SDS-PAGE, which serve as loading controls. B and C, respiration measured in the presence of 2 mm NADH. n = 6–9. B, phosphate/oxygen ratios. C, respiratory control ratios. D, representative TMRM time traces of mitochondria isolated from the indicated yeast strains following the addition of 2 mm NADH (to establish the Δψ_m) and two sequential additions of 45 μm ADP to induce phosphorylation cycles, manifest as transient depolarizations. E, average times required for the re-establishment of maximal (state 4) Δψ_m following ADP addition for the yeast strains indicated. *, p < 0.05; ***, p < 0.001.

FIGURE 6.

Mitochondrial proton leak is increased in the absence of CL. A, respiration of isolated mitochondria measured in the presence of NADH (state 4) or NADH and ADP (state 3). ***, p < 0.001. B, relative membrane potentials from five independent experiments were plotted (circles). The mean ± S.E. are displayed as black bars. C, membrane potential of the indicated strains measured in the presence of oligomycin (left), carboxyatractyloside (middle), or oligomycin + carboxyatractyloside (right).

FIGURE 7.

Individual components of OXPHOS are not affected by unremodeled CL. A, uncoupled respiration measured in the presence of 2 mm NADH and 10 μm CCCP. B, complex III; C, complex IV activity, measured in n-dodecyl β-d-maltoside mitochondrial extracts. **, p < 0.01. D, mitochondrial proteins from the indicated strains were separated by SDS-PAGE and immunoblotted. * indicates a nonspecific cross-reaction of the Cld1p antisera.