Ethanolamine ameliorates mitochondrial dysfunction in cardiolipin-deficient yeast cells

Abstract

Cardiolipin (CL) is a signature phospholipid of the mitochondria required for the formation of mitochondrial respiratory chain (MRC) supercomplexes. The destabilization of MRC supercomplexes is the proximal cause of the pathology associated with the depletion of CL in patients with Barth syndrome. Thus, promoting supercomplex formation could ameliorate mitochondrial dysfunction associated with CL depletion. However, to date, physiologically relevant small-molecule regulators of supercomplex formation have not been identified. Here, we report that ethanolamine (Etn) supplementation rescues the MRC defects by promoting supercomplex assembly in a yeast model of Barth syndrome. We discovered this novel role of Etn while testing the hypothesis that elevating mitochondrial phosphatidylethanolamine (PE), a phospholipid suggested to overlap in function with CL, could compensate for CL deficiency. We found that the Etn supplementation rescues the respiratory growth of CL-deficient Saccharomyces cerevisiae cells in a dose-dependent manner but independently of its incorporation into PE. The rescue was specifically dependent on Etn but not choline or serine, the other phospholipid precursors. Etn improved mitochondrial function by restoring the expression of MRC proteins and promoting supercomplex assembly in CL-deficient cells. Consistent with this mechanism, overexpression of Cox4, the MRC complex IV subunit, was sufficient to promote supercomplex formation in CL-deficient cells. Taken together, our work identifies a novel role of a ubiquitous metabolite, Etn, in attenuating mitochondrial dysfunction caused by CL deficiency.

Keywords: Barth syndrome; cardiolipin; cytochrome c oxidase (Complex IV); ethanolamine; mitochondria; mitochondrial respiratory chain complex; phospholipid; respiratory supercomplexes.

Figure 1.

Biochemical pathways for PE, PC, and CL biosynthesis in the yeast S. cerevisiae. CL biosynthesis occurs exclusively in the mitochondria, where Crd1 synthesizes nascent CL_p from PG. The resulting CL_p is deacylated by the phospholipase Cld1 to produce monolysocardiolipin (MLCL), which is then reacylated by the transacylase Taz1 to form mature cardiolipin (CL_m). Mutations in the human homologue of TAZ1 result in Barth syndrome. CL biosynthesis depends on the import of PA from endoplasmic reticulum, which is converted to CDP-DAG by Tam41. Pgs1 catalyzes conversion of CDP-DAG to PGP, which is then dephosphorylated to PG by Gep4. PE biosynthesis in yeast can occur by the following: 1) Psd1-catalyzed decarboxylation of PS in the mitochondria; 2) incorporation of Etn via the cytosolic/endoplasmic reticulum Kennedy pathway enzymes Eki1, Ect1, and Ept1, respectively; and 3) the acylation of lyso-PE by Ale1. The Kennedy pathway enzymes Cki1, Pct1, and Cpt1 can utilize choline to biosynthesize PC, a bilayer-forming phospholipid that is imported into mitochondria. CL_p, precursor cardiolipin; CL_m, mature cardiolipin; PG, phosphatidylglycerol; PGP, phosphatidylglycerol phosphate; CDP, cytidine diphosphate; DAG, diacylglycerol.

Figure 2.

Ethanolamine supplementation rescues respiratory growth of CL-deficient yeast cells. A, 10-fold serial dilutions of WT, taz1Δ, and crd1Δ cells were seeded on to the SC glucose, SC ethanol, and SC ethanol + 2 mm Etn media, and images were captured after 2 days of growth on SC glucose, 5 days on SC ethanol ± 2 mm Etn at 30 °C (upper panel), and 7 days on SC ethanol ± 2 mm Etn at 37 °C (lower panel). Data are representative of at least three independent experiments. B, growth of taz1Δ and crd1Δ cells in SC ethanol with the indicated concentrations of Etn (0–5 mm) was monitored by measuring absorbance at 600 nm at 72 or 48 h for taz1Δ cells and crd1Δ cells, respectively, and EC₅₀ values for Etn were calculated using GraphPad Prism. Data are expressed as mean ± S.D. (n = 3). For growth comparison, WT cells were cultured in SC ethanol medium without Etn supplementation for the indicated temperatures and time periods. C, growth of BY4741 WT, taz1Δ, and crd1Δ cells in SC glucose, SC ethanol, and SC ethanol + 2 mm Etn media at 30 °C (upper panel) and 37 °C (lower panel) was monitored by measuring absorbance at 600 nm. Data are representative of at least three independent measurements. D, mitochondrial phospholipid composition of WT cells grown in SC ethanol and taz1Δ and crd1Δ cells grown in SC ethanol ± Etn. Data are expressed as mean ± S.D. (n = 3); **, p < 0.005; *, p < 0.05; NS, not significant. PI, phosphatidylinositol; PG, phosphatidylglycerol.

Figure 3.

Ethanolamine-mediated rescue of respiratory growth of CL-deficient cells is independent of PE biosynthesis. A, 10-fold serial dilutions of WT, ect1Δ, taz1Δ, and ect1Δtaz1Δ cells were seeded on to the indicated media, and images were captured after 2 days of growth on SC glucose and 7 days on SC ethanol ± 2 mm Etn at 37 °C. Data are representative of at least three independent experiments. B, total cellular PE levels of the indicated strains grown in SC ethanol ± 2 mm Etn. Data are expressed as mean ± S.D. (n = 3); *, p < 0.05, NS, not significant. C, 10-fold serial dilutions of WT, eki1Δcki1Δ, taz1Δ, eki1Δcki1Δtaz1Δ. D, WT, hnm1Δ, taz1Δ, and hnm1Δtaz1Δ cells were seeded onto the indicated media, and images were captured after 2 days of growth on SC glucose and 7 days on SC ethanol ± 2 mm Etn at 37 °C. Data are representative of at least three independent experiments. E, box plots of relative intracellular Etn abundance from WT, hnm1Δ, taz1Δ, and hnm1Δtaz1Δ grown in SC ethanol ± Etn. Etn levels were measured by LC-MS and expressed as peak intensity in arbitrary units (A.U.). Box plots show individual data points, median, first, and third quartiles, and greatest values within 1.5 inter-quartile range. Data are representative of at least three independent experiments. Data are expressed as mean ± S.D. (n = 3). F, WT and hnm1Δ cells were labeled with 1 μCi of [¹⁴C]Etn for 24 h, and radioactive Etn counts were measured in cpm (CPM) and expressed as % of WT. Data are expressed as mean ± S.D. (n = 3); ***, p < 0.001. G, growth of taz1Δhnm1Δ cells in SC ethanol with indicated concentrations of Etn (0–10 mm) was monitored by measuring absorbance at 600 nm at 72 h, and EC₅₀ values for Etn were calculated using GraphPad Prism. Data are expressed as mean ± S.D. (n = 3). H, WT cells, grown in presence or absence of 10 mm choline, were radiolabeled with 1 μCi of [¹⁴C]Etn for 24 h, and intracellular radioactivity was measured and expressed as % of WT intensity. Data are expressed as mean ± S.D. (n = 3); ***, p < 0.001. I, 10-fold serial dilutions of WT, taz1Δ, and crd1Δ cells were seeded onto the indicated media, and images were captured after 2 days of growth on SC glucose and 7 days on SC ethanol ± Cho at 37 °C. Data are representative of at least three independent experiments.

Figure 4.

Respiratory growth of CL-deficient cells is rescued by ethanolamine analogues propanolamine and monomethylethanolamine. A, chemical structures of Etn, MME, DME, Cho, Ser, and Prn. B, growth of WT and taz1Δ cells at 37 °C; C, WT and crd1Δ cells at 30 °C in SC ethanol (SCE), with the indicated supplementations. Growth was monitored by measuring absorbance at 600 nm and presented as a heat map. Data are average of five independent measurements.

Figure 5.

Ethanolamine supplementation partially restores supercomplex formation in taz1Δ cells. A, mitochondria from WT cells grown in SC ethanol and taz1Δ cells grown in SC ethanol ± 2 mm Etn were solubilized by 1% digitonin and subjected to BN-PAGE/Western blot analysis. Complexes containing MRC complex III were detected by anti-Rip1 antibody. B, relative abundance of MRC complex III containing complexes from A were quantified by densitometric analysis. Data are expressed as mean ± S.D. (n = 4). C, samples from A were probed with anti-Cox2 antibody to detect MRC complex IV containing supercomplexes and complex IV monomer. D, relative abundance of MRC complex IV containing complexes from C were quantified by densitometric analysis. Data are expressed as mean ± S.D. (n = 3). E, mitochondria from WT, taz1Δ, and ect1Δtaz1Δ cells grown in SC ethanol ± 2 mm Etn were solubilized by 1% digitonin and subjected to BN-PAGE/Western blot analysis and MRC complex III; F, complex IV was detected by probing with anti-Rip1 and anti-Cox2 antibodies, respectively. III₂IV₂, large supercomplex; III₂IV, small supercomplex; III₂, complex III dimer; IV, complex IV monomer.

Figure 6.

Ethanolamine supplementation restores MRC complex IV levels and activity in crd1Δ cells. A, mitochondria from WT cells grown in SC ethanol and crd1Δ cells grown in SC ethanol ± 2 mm Etn were solubilized by 1% digitonin and subjected to BN-PAGE/Western blot analysis. MRC complex III containing complexes were detected by anti-Rip1 antibody. B, relative abundance of MRC complex III containing complexes from A were quantified by densitometric analysis. Data are expressed as mean ± S.D. (n = 3). C, samples from A were probed with anti-Cox2 antibody to detect MRC complex IV containing supercomplexes and complex IV monomer. D, relative abundance of MRC complex IV containing complexes from C were quantified by densitometric analysis. Data are expressed as mean ± S.D. (n = 3). E, MRC subunits of complexes II (Sdh1), III (Cor1 and Qcr2), and IV (Cox2 and Cox4) were analyzed by SDS-PAGE/Western blotting. Mitochondrial proteins aconitase and porin were used as a loading control. Data are representative of at least three independent experiments. F, digitonin-solubilized MRC complexes from WT and crd1Δ cells were separated by CN-PAGE, followed by in-gel activity staining for complex IV. In-gel activity of complex IV was quantified by densitometric analysis, and relative activity was plotted. Data were normalized to WT cells and expressed as mean ± S.D. (n = 3); *, p < 0.05.

Figure 7.

Cox4 overexpression rescues mitochondrial respiratory chain supercomplex assembly in cardiolipin-deficient cells. A, mitochondria from WT, taz1Δ, and crd1Δ cells, with or without Cox4 overexpression, were subjected to SDS-PAGE/Western blotting. B, mitochondria from WT, taz1Δ, and crd1Δ cells, with or without Cox4 overexpression, were solubilized by 1% digitonin and subjected to BN-PAGE/Western blot analysis. MRC complex III containing complexes were detected by anti-Rip1. C, MRC complex IV containing complexes were detected by anti-Cox2 antibodies, respectively. Data are representative of three independent measurements.

Figure 8.

Ethanolamine supplementation reduces protein carbonylation in CL-deficient cells. WT, taz1Δ, and crd1Δ (A) or WT, ect1Δ, ect1Δtaz1Δ, and ect1Δcrd1Δ cells (B) were grown in SC ethanol ± 2 mm Etn to early stationary phase; protein was extracted, and protein carbonylation was measured as described under “Experimental procedures.” Data are expressed as mean ± S.D. (n = 3); **, p < 0.005; *, p < 0.05.

Figure 9.

Ethanolamine-mediated rescue is specific to CL deficiency. A, growth of WT, rcf1Δ, and rcf2Δ cells in SC glucose and SC ethanol ± 2 mm Etn media was monitored at 30 °C (upper panel) and 37 °C (lower panel) by measuring absorbance at 600 nm. Data are representative of at least two independent measurements. B, 10-fold serial dilutions of WT, taz1Δ, sdh2Δ, bcs1Δ, shy1Δ, and atp12Δ cells were seeded onto the indicated media, and images were captured after 2 days of growth on SC glucose and 5 days on SC ethanol ± 2 mm Etn at 30 °C. Data are representative of at least three independent trials. C, mitochondria from YP galactose grown WT, sdh2Δ, bcs1Δ, and shy1Δ cells were subjected to Western blot analysis. Mitochondrial respiratory chain complexes II, III, and IV subunits were probed using anti-Sdh2, anti-Rip1, and anti-Cox2 antibodies, respectively. Porin was used as a loading control.