Loss of Cardiolipin Leads to Perturbation of Acetyl-CoA Synthesis

Abstract

Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, plays an important role in mitochondrial processes and bioenergetics. CL is synthesized de novo and undergoes remodeling in the mitochondrial membranes. Perturbation of CL remodeling leads to the rare X-linked genetic disorder Barth syndrome, which shows disparities in clinical presentation. To uncover biochemical modifiers that exacerbate CL deficiency, we carried out a synthetic genetic array screen to identify synthetic lethal interactions with the yeast CL synthase mutant crd1Δ. The results indicated that crd1Δ is synthetically lethal with mutants in pyruvate dehydrogenase (PDH), which catalyzes the conversion of pyruvate to acetyl-CoA. Acetyl-CoA levels were decreased in the mutant. The synthesis of acetyl-CoA depends primarily on the PDH-catalyzed conversion of pyruvate in the mitochondria and on the PDH bypass in the cytosol, which synthesizes acetyl-CoA from acetate. Consistent with perturbation of the PDH bypass, crd1Δ cells grown on acetate as the sole carbon source exhibited decreased growth, decreased acetyl-CoA, and increased intracellular acetate levels resulting from decreased acetyl-CoA synthetase activity. PDH mRNA and protein levels were up-regulated in crd1Δ cells, but PDH enzyme activity was not increased, indicating that PDH up-regulation did not compensate for defects in the PDH bypass. These findings demonstrate for the first time that CL is required for acetyl-CoA synthesis, which is decreased in CL-deficient cells as a result of a defective PDH bypass pathway.

Keywords: acetyl coenzyme A (acetyl-CoA); cardiolipin; energy metabolism; pyruvate dehydrogenase complex (PDC); yeast genetics.

FIGURE 1.

Functional classification of genes exhibiting synthetic lethality with crd1Δ. A, synthetic lethal interactions identified by the SGA screen were classified by biological process based on gene ontology. B, synthetic lethal partners of crd1Δ identified eight biological processes. Genes that are functionally related radiate from a central point.

FIGURE 2.

Genetic interaction of PDH mutants with crd1Δ. A, cells were pre-cultured in YPD overnight, diluted, plated in equal number on YNBD, and incubated at 30 or 37 °C for 3–5 days. B, synthetic interaction between CRD1 and PDH mutants was determined by examining growth of the double mutant compared with isogenic parental strains and wild type.

FIGURE 3.

Decreased acetyl-CoA levels in crd1Δ cells. Wild-type and crd1Δ cells were grown at the indicated temperatures in YPD until the cells reached an A₅₅₀ of 1. Cells were pelleted, and acetyl-CoA was extracted using glutaryl-CoA as an internal standard. The clarified extract (10 μl) was analyzed by HPLC-MS/MS. Data shown are mean ± S.D. (n = 6) (*, p < 0.05; **, p < 0.01).

FIGURE 4.

Decreased growth of crd1Δ cells on acetate. A, cells were pre-cultured overnight in YNBD and diluted, plated in equal number on YNBD and YNBA, and incubated at 30 °C for 3–5 days. B, growth in liquid YNBA was determined by measuring A₅₅₀. The growth curve values are an average of at least three experiments ± S.D., n ≥3.

FIGURE 5.

Increased acetate levels in crd1Δ cells. Wild-type and crd1Δ cells were grown in YNBA at 30 °C, and intracellular acetate levels were determined as described under “Experimental Procedures.” Data shown are mean ± S.D. (n = 6) (***, p < 0.001).

FIGURE 6.

Perturbation of PDH bypass in crd1Δ cells. Wild-type and crd1Δ cells were grown in YNBA at 30 °C to the logarithmic phase. A, acetyl-CoA levels were determined as described under “Experimental Procedures.” Data shown are mean ± S.D. (n = 6) (**, p < 0.01). B, enzyme activity (units/mg) of acetyl-CoA synthetase was assayed as described under “Experimental Procedures.” Data shown are mean ± S.D. (n = 6) (***, p < 0.001).

FIGURE 7.

PDH activity is not increased in crd1Δ cells. Cells were pre-cultured in YPD overnight, diluted, plated in equal numbers on YNBD, and incubated at 30 or 37 °C for 3–5 days. A, mRNA levels of PDH genes from cells grown in YPD at 30 °C to the logarithmic phase were quantified by qPCR. Values are reported as fold change in expression compared with wild type. Expression was normalized to the mRNA levels of the internal control ACT1. Data shown are mean ± S.D. (n = 6) (*, p < 0.05; **, p < 0.01; ***, p < 0.001). B, anti-HA antibody was used to detect HA-tagged proteins in cell lysates by Western blotting analysis. 50 μg of total protein was loaded for each sample, and α-tubulin was used as an internal control. The molecular masses are as follows: tubulin, 50 kDa; PDA1, 39.4 kDa; PDB1, 43.3 kDa; LAT1, 69 kDa; LPD1, 54 kDa; and PDX1, 37 kDa (top). The levels of PDH protein were quantified using ImageJ software (bottom). Data shown are mean ± S.D. (n = 3) (*, p < 0.05; **, p < 0.01). C, enzyme activity (units/mg) of PDH was assayed in cell extracts as described under “Experimental Procedures.” Data shown are mean ± S.D. (n = 6). D, enzyme activity (units/mg) of PDH was assayed in the presence or absence of CL as described under “Experimental Procedures.” Data shown are mean ± S.D. (n = 6) (*, p < 0.05; ***, p < 0.001).

FIGURE 8.

Model, perturbation of acetyl-CoA synthesis in crd1Δ cells. In the proposed model, loss of CL leads to decreased acetyl-CoA synthetase enzyme activity, resulting in accumulation of acetate and decreased synthesis of acetyl-CoA by the PDH bypass. To compensate for defects in the PDH bypass, PDH gene expression and protein synthesis are increased in CL-deficient cells. However, PDH activity is not increased, possibly because mitochondrial import, enzymatic activity, or assembly of PDH inside the mitochondria requires CL. Thus, a defective PDH bypass pathway results in decreased synthesis of acetyl-CoA, which is not compensated by PDH.