Loss of the mitochondrial lipid cardiolipin leads to decreased glutathione synthesis

Abstract

Previous studies demonstrated that loss of CL in the yeast mutant crd1Δ leads to perturbation of mitochondrial iron-sulfur (Fe–S) cluster biogenesis, resulting in decreased activity of mitochondrial and cytosolic Fe-S-requiring enzymes, including aconitase and sulfite reductase. In the current study, we show that crd1Δ cells exhibit decreased levels of glutamate and cysteine and are deficient in the essential antioxidant, glutathione, a tripeptide of glutamate, cysteine, and glycine. Glutathione is the most abundant non-protein thiol essential for maintaining intracellular redox potential in almost all eukaryotes, including yeast. Consistent with glutathione deficiency, the growth defect of crd1Δ cells at elevated temperature was rescued by supplementation of glutathione or glutamate and cysteine. Sensitivity to the oxidants iron (FeSO₄) and hydrogen peroxide (H₂O₂), was rescued by supplementation of glutathione. The decreased intracellular glutathione concentration in crd1Δ was restored by supplementation of glutamate and cysteine, but not by overexpressing YAP1, an activator of expression of glutathione biosynthetic enzymes. These findings show for the first time that CL plays a critical role in regulating intracellular glutathione metabolism.

1. Introduction

Cardiolipin (CL) is a unique dimeric phospholipid that is almost exclusively present in mitochondrial membranes and plays a key role in mitochondrial function. Perturbation of CL metabolism in humans leads to Barth syndrome (BTHS), a severe genetic disorder that results in cardio- and skeletal myopathy, neutropenia, and numerous metabolic abnormalities [3]. Although the defect in BTHS is known to result from mutations in the CL remodeling enzyme tafazzin [4], the link between CL deficiency and the pathology in BTHS is not understood. Recent findings indicate that metabolic dysregulation is an important mechanism underlying the pathology in BTHS [5,6]. Studies using yeast CL mutants have provided insight into the role of CL in mitochondrial function and energy metabolism. In addition to the now well-established role of CL in oxidative phosphorylation [7–9], more recent studies using the yeast model have determined that CL is required for acetyl-CoA synthesis [10] and for the biogenesis of Fe–S clusters [11], which are incorporated as co-factors into iron-dependent enzymes [14]. These include enzymes required for mitochondrial bioenergetics, the TCA cycle, and amino acid biosynthesis. Among the latter are the enzymes aconitase (Aco1), glutamate synthase (Glt1), sulfite reductase (two subunits each of Met5 and Met10), dihydroxyacid dehydratase (Ilv3), isopropylmalate isomerase (Leu1), and homoaconitase (Lys4) [15]. Therefore, decreased Fe–S biogenesis in the mitochondria is expected to disrupt amino acid synthesis, leading to severe metabolic perturbation.

The yeast CL synthase mutant crd1Δ, which cannot synthesize CL, exhibits decreased activity of mitochondrial and cytosolic Fe–S enzymes, including aconitase and sulfite reductase, among others [11]. Aconitase catalyzes the conversion of citrate to isocitrate, which is subsequently decarboxylated to α-ketoglutarate by isocitrate dehydrogenase [16,17]. In addition to its role in the TCA cycle, α-ketoglutarate is a substrate for the synthesis of glutamate [19]. In the sulfur biosynthetic pathway, sulfate taken up from the surroundings is converted to sulfite and subsequently reduced by sulfite reductase to sulfide, which combines with O-acetylhomoserine to form homocysteine [20]. Homocysteine can be interconverted to methionine or to cystathionine, which is used to generate cysteine [20–22]. Deficiencies in Fe–S enzymes aconitase and sulfite reductase lead to auxotrophy for glutamate and cysteine/methionine, respectively [23,24].

Depletion of glutamate and cysteine is expected to decrease the synthesis of glutathione, a tripeptide of cysteine, glutamate, and glycine [25–27]. The synthesis of glutathione is initiated by the conversion of glutamate and cysteine to γ-glutamylcysteine, catalyzed by Gsh1 (glutamate-cysteine ligase). Gsh2 (glutathione synthase) then catalyzes the ligation of glycine to the dipeptide [28,29]. Deletion of GSH1 leads to glutathione auxotrophy, while cells lacking GSH2 are able to grow in minimal medium lacking glutathione [30,31]. Expression of GSH1 and GSH2 in response to oxidative stress is under the regulatory control of the transcription factor Yap1 [32–35]. Activation of Yap1 by oxidative stress and temperature shock, or by overexpression of the YAP1 gene, augments the expression of GSH1 and GSH2 and elevates GSH levels [33–35].

Glutathione is the most abundant intracellular antioxidant that plays a critical role in maintaining redox potential and protects the intracellular environment against reactive oxygen species (ROS), xenobiotics, heavy metals, and temperature-mediated stress [36–39]. Glutathione acts as a co-factor for antioxidant enzymes such as glutathione peroxidase, which utilize glutathione to neutralize ROS, including hydrogen peroxide (H₂O₂) [31]. The antioxidant function of glutathione depends on the active thiol group (-SH) [31]. Glutathione donates electrons necessary to reduce the ROS and, in turn, is oxidized to glutathione disulfide (GSSG). Depletion of glutathione leads to increased sensitivity to oxidants such as H₂O₂, while disruption of GSH synthesis by deletion of GSH1 leads to growth arrest in synthetic medium lacking GSH, cysteine, or reducing agent, dithiothreitol (DTT) [28–31]. Therefore, to maintain vital cellular functions, intracellular glutathione levels must be rigorously maintained, either by de novo synthesis or uptake from the growth medium.

In this study, we show that CL-deficient crd1Δ cells exhibit decreased levels of glutamate and cysteine and decreased synthesis of glutathione. Glutathione levels, as well as defective growth in response to elevated temperature, are rescued by supplementation of glutamate and cysteine. Sensitivity to the oxidants iron (FeSO₄) and hydrogen peroxide (H₂O₂), was rescued by supplementation of glutathione. These findings suggest that decreased activities of Fe-S-requiring enzymes in CL-deficient cells result in defects in the glutamate and cysteine biosynthetic pathways, leading to deficiencies in these amino acids and decreased synthesis of glutathione. This is the first demonstration of a link between the mitochondrial lipid CL and synthesis of the antioxidant glutathione.

2. Materials and methods

2.1. Yeast strains and growth media

The yeast strains used in this study are listed in Table 1. Synthetic defined (SD) medium contained adenine (20.25 mg/L), arginine (20 mg/L), histidine (20 mg/L), leucine (60 mg/L), lysine (20 mg/L), methionine (20 mg/L), threonine (300 mg/L), tryptophan (20 mg/L), uracil (20 mg/L), yeast nitrogen base without amino acids (Difco, Detroit, MI), and either (fermentative) glucose (2%) or (respiratory-fermentative) galactose (2%) as a carbon source. SD dropout medium contained all of the above-mentioned ingredients except for the indicated amino acid. For growth experiments on excess iron, 1 μM CuSO₄ was used, and FeSO₄ was solubilized in 0.1 N HCl, filter-sterilized, and added to the culture medium at the indicated concentration. GSH was prepared in distilled water, filter-sterilized, and added to the culture medium at the indicated concentration.

Table 1.

Yeast strains and plasmids used in this study.

Strain or plasmid	Genotype	Source or reference
FGY3	MATa, ura3–52, lys2–801, ade2–101, trp1-Δ1, his3-Δ200, leu2-Δ1	[1]
FGY2	MATa, ura3–52, lys2–801, ade2–101, trp1-Δ1, his3-Δ200, leu2-Δ1, crd1Δ::URA3	[1]
BY4742	MATα, his 3Δ1, leu 2Δ0, lys 2Δ0, ura 3Δ0	Invitrogen
VGY1	MATα, his 301, leu 200, lys 200, ura 300, crd1Δ::URA3	[2]
gsh1Δ	MATa, his 301, leu 200, met 1500, ura 300, gsh1Δ::KanMX4	Invitrogen
VGY1gsh1Δ	MATa, his 301, leu 200, met 1500, ura 300, gsh1Δ::KanMX4, crd1Δ::URA3	This study
YEp351	High-copy number plasmid, LEU2 nutritional marker	[12]
pRS415	Centromere plasmid (low/single copy), LEU2 nutritional marker	[13]
YEp351-YAP1	Derivative of YEp351, expresses YAP1 from the native promoter	[18]

2.2. Biochemical assays

Cell extracts were prepared by resuspending cells in 500 μL of TNTEG buffer (10 mM Tris-Cl pH 7.4, 2.5 mM EDTA, 150 mM NaCl, 10% (vol/vol) glycerol, 0.5% (vol/vol) Triton X-100) and subjecting them to mechanical breakage with glass beads. Cell debris and unbroken cells were separated by low speed centrifugation (2000g for 5 min at 4 °C). The obtained supernatant was further centrifuged at 13,000 g for 10 min and the resulting supernatant was transferred to a new tube. Total protein concentration was determined with a Bradford assay kit (BioRad) using BSA as the standard. Enzyme assays were performed in whole-cell extracts as follows: Aconitase was assayed by the aconitase-isocitrate dehydrogenase coupled assay in which NADPH formation was monitored at A₃₄₀ [40]. Isopropylmalate isomerase was assayed by monitoring formation of the UV-absorbing double bond of isopropylmalate at A₂₃₅ due to dehydration of 3-isopropylmalate [40]. For the isopropylmalate isomerase assay, because the parental strains carry the leu2Δ null mutation, they were transformed with a low/single copy pRS415 plasmid that expresses LEU2. Statistical signficance for both assays was determined by two-tailed Student’s t-test.

2.3. Amino acid analysis

Amino acid analyses were performed at the Protein Chemistry Laboratory, Texas A&M University, College Station, Texas. Total cell extracts of WT and crd1Δ cells, standards, and control were passed through a 5000 MWCU filter (Vivaspin 500) to remove cellular proteins and large peptides. The filtrates (in duplicates) were combined with internal standards [normaline (Sigma #N7502) and sarcosine (Sigma #S7672)] and dithiodipropionic acid (100 mM) to convert cysteine and cystine to Cys MPA (S-(-2-carbpxythethylthio-Cys)). This mixture was heated to 150 °C for 45 min with vapor-phase 6 N HCl. Cys-MPA in all standards, controls, and samples was quantified using an Agilent 1260 HPLC analyzer. Alanine levels in the standards, control, and samples were quantified/normalized to the cysteine levels.

Agilent 1260 HPLC analysis was performed by pre-column derivatization of the amino acids with o-phthalaldehyde (OPA, Agilent #5061–3335) and 9-fluoromethyl-chloroformate (FMOC, Agilent #5061–3337) after addition of 0.4 N borate buffer (Agilent #5061–3339) to bring to pH 10 for optimum derivatization. OPA reacts with primary amino acids and FMOC with secondary amino acids (proline). Both reagents react rapidly and quantitatively and give highly UV-absorbing isoindole derivatives. The derivatized amino acids were separated by reverse phase HPLC and detected by UV absorbance (primary at 338/390 nm and secondary at 266/324 nm) with a variable wavelength detector (G1365D). The assay was calibrated by a standard (Agilent #5061–3331), which undergoes the same treatment as the samples and control.

2.4. Glutathione levels

Total cell extracts from WT and crd1Δ cells grown at 35 °C to log phase were deproteinized with 5% 5-sulfosalicylic acid solution, centrifuged to remove precipitated proteins, and then assayed for total glutathione (GSH + GSSG). Total glutathione concentrations were measured using a kinetic assay (Sigma-Aldrich #CS0260) in which catalytic amounts (nmoles) of GSH cause a continuous reduction of 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) to TNB, and the GSSG formed is recycled by glutathione reductase and NADPH. Statistical significance was determined by two-tailed paired Student’s t-test.

3. Results

3.1. Decreased glutamate and cysteine in crd1Δ cells

To test the prediction that deficiencies in mitochondrial and cytosolic Fe–S enzymes in crd1Δ cells lead to glutamate and cysteine auxotrophies that contribute to growth defects, the effect of supplementation of these amino acids on growth of the CL mutant at elevated temperature was determined. Supplementation with cysteine (0.03%) and glutamate (0.01%) together restored growth of the mutant at elevated temperature to wild type levels (Fig. 1A). Cysteine alone partially rescued growth of crd1Δ (Fig. 1C), but glutamate alone did not (Fig. 1B). This is consistent with previous reports suggesting that cysteine is the limiting substrate for intracellular glutathione synthesis [25,41]. The growth phenotype of crd1Δ is similar to that of gsh1Δ, which is unable to grow in minimal medium in the absence of GSH, cysteine, or DTT. Free amino acid levels were assayed to determine if the growth defect of crd1Δ at elevated temperature reflected deficiencies in intracellular glutamate and cysteine. HPLC analysis indicated that the intracellular levels of glutamate and cysteine are reduced in crd1Δ cells by 35.8% and 21.21%, respectively (Table 2). In addition, CL-deficient cells showed decreased levels of leucine, aspartate, glutamine, and proline, and elevated levels of serine, histidine, threonine, alanine, valine, methionine, isoleucine, and lysine. While the cellular consequences that result in elevated levels of multiple amino acids in the CL mutant are not clear, decreased levels of aspartate, glutamine, and proline are likely due to defective Fe–S biogenesis. Our previous study has shown that the CL mutant exhibits decreased activity of Aco1 [11], which catalyzes the conversion of citrate to isocitrate, a critical intermediate of the TCA cycle. Isocitrate is converted to α-ketoglutarate, a key intermediate for glutamate biosynthesis, which can be subsequently interconverted to glutamine and proline. Oxaloacetate, another intermediate of the TCA cycle, is essential for aspartate biosynthesis. As the parental strains carry the leu2Δ null mutation, growth medium was supplemented with leucine (60 mg/L). Decreased leucine levels in crd1Δ suggest increased catabolism or decreased uptake, however the underlying cause for this is unclear.

Fig. 1.

Supplementation of glutamate and cysteine restores the growth of crd1Δ at elevated temperature. Cells were precultured in SD galactose overnight, serially diluted, and spotted on SD galactose plates containing the indicated amounts of either glutamate and cysteine together (A), glutamate alone (B), or cysteine alone (C). Plates were incubated at 37 °C for 4 days before images were taken.

Table 2.

Free amino acid analysis in CL mutant crd1Δ. Cells were precultured in YPD overnight at 30 °C, cells were rinsed in water and inoculated in synthetic galactose medium lacking glutamate and methionine/cysteine at 35 °C and grown to the logarithmic phase. Cells were then rinsed 3 times in PBS (pH 7.4) to remove residual medium, and total amino acid levels were determined by HPLC. Data shown are mean ± S.D. (n = 4).

Amino acid	WT nmol (SD)	crd1Δ nmol (SD)
ASP	2.6 ± 0.85	1.2 ± 0
GLU	25.3 ± 3.39	16.25 ± 0.07
CYS	1.65 ± 0.21	1.3 ± 0
SER	1 ± 0	1.25 ± 0.07
GLN	9.55 ± 0.49	3.8 ± 0.57
HIS	2.1 ± 0	2.65 ± 0.07
GLY	1.65 ± 0.35	2.45 ± 0.07
THR	32.05 ± 0.64	41.5 ± 1.56
ALA	5.35 ± 0.07	6.15 ± 0.07
ARG	6.3 ± 0.85	6.65 ± 0.21
TYR	0.65 ± 0.21	0.65 ± 0.07
VAL	1.2 ± 0.14	1.75 ± 0.07
MET	0.3 ± 0	0.4 ± 0
TRP	0.35 ± 0.07	0.3 ± 0.14
PHE	0.35 ± 0.07	0.35 ± 0.07
ILE	2.85 ± 0.35	4.7 ± 0
LEU	1.2 ± 0.28	0.2 ± 0
LYS	5.35 ± 0.21	8.45 ± 0.49
PRO	1.7 ± 0.14	1.05 ± 0.07

SD = standard deviation.

3.2. Decreased glutathione in crd1Δ cells

Because glutamate and cysteine are essential precursors for the synthesis of glutathione, we predicted that deficiencies in these amino acids would lead to decreased glutathione. In agreement with this, total cellular glutathione levels [GSH (reduced) + GSSG (oxidized)] were decreased by 35.74 ± 7.57% in crd1Δ cells compared to WT (Fig. 2A). Supplementation of both glutamate and cysteine increased intracellular glutathione to near-wild type levels. Growth of crd1Δ cells at elevated temperature was restored by glutathione supplementation (Fig. 2B). The glutathione mutant, gsh1Δ, does not exhibit a growth deficiency in complex fermentable medium (e.g. YPD); however, the mutant exhibits growth arrest in synthetic medium lacking glutathione, cysteine, or DTT [30,32,42]. This suggests that an intracellular reducing environment is essential for the growth of gsh1Δ in synthetic fermentable medium. Based on this, we examined how deletion of GSH1 may affect the growth of crd1Δ. We noted that perturbation of GSH synthesis further exacerbated the growth of crd1Δ cells, such that crd1Δ exhibited lethality with gsh1Δ in synthetic fermentable medium (Fig. 2C).

Fig. 2.

Cardiolipin-deficient cells exhibit decreased glutathione levels, and growth of crd1Δ at elevated temperature is rescued by supplementation with GSH. (A) crd1Δ cells show decreased levels of glutathione (GSH + GSSG). Cells were precultured in YPD overnight at 30 °C, washed with distilled water and inoculated in SD galactose medium lacking glutamate and methionine/cysteine at 35 °C, and grown to the logarithmic phase, either in the presence or absence of glutamate and cysteine. Total glutathione levels were measured as described in materials and methods. Error bars represent mean ± SD (n = 6, two-tailed paired Student’s t-test, *p < 0.005). (B) GSH supplementation rescued growth of crd1Δ. Cells were precultured in SD galactose overnight, serially diluted, spotted on SD galactose plates containing the indicated amounts of GSH, and incubated at 37 °C for 4 days. (C) crd1Δ exhibits synthetic lethality with gsh1Δ in fermentable medium. Cells were precultured in YPD overnight, washed with distilled water, serially diluted, and spotted on YPD (left-panel) or SD glucose (right-panel), and incubated at 30 °C. (D) Overexpression of YAP1 does not rescue growth of crd1Δ. Cells were precultured in SD galactose overnight, serially diluted, spotted on SD galactose plates lacking leucine, and incubated at 30 °C (left-panel) and 36 °C (right-panel) for 4–5 days.

If glutathione deficiency in crd1Δ is due to decreased glutathione biosynthetic enzymes, the deficiency might be rescued by overexpression of YAP1, which encodes the transcription factor that regulates the expression of GSH1 and GSH2 [28,29,33]. GSH1 encodes glutamate-cysteine ligase, a rate-limiting enzyme that catalyzes the formation of γ-glutamylcysteine from glutamic acid and cysteine; GSH2 encodes glutathione synthase, which catalyzes the ligation of glycine with the dipeptide, producing glutathione. Data in Fig. 2D show that overexpression of YAP1 did not rescue the growth of crd1Δ in galactose medium (respiratory-fermentative) at elevated temperature, suggesting that the decrease in glutathione levels in crd1Δ is most likely not due to deficiency of glutathione biosynthetic enzymes.

3.3. Supplementation with glutathione rescued the growth defect of CL-mutant cells in the presence of oxidants

The gsh1Δ mutant exhibits hypersensitivity to oxidants, including H₂O₂ [30,43]. This phenotype is also characteristic of CL-deficient cells, which exhibit increased oxidative stress in respiratory (ethanol) and respiratory-fermentative (galactose) growth conditions, and are sensitive to ethanol, iron, and H₂O₂ [11,44]. To address the possibility that glutathione deficiency in crd1Δ cells may account for sensitivity to oxidants, the effect of glutathione supplementation on growth of crd1Δ cells in the presence of FeSO₄ or H₂O₂ was determined. Glutathione supplementation significantly improved growth of crd1Δ cells in the presence of iron and H₂O₂ (Fig. 3).

Fig. 3.

Iron and hydrogen peroxide sensitivity in crd1Δ is rescued by GSH. Cells were precultured in YPD overnight, washed with distilled water, serially diluted, and spotted on SD galactose plates containing (A) 0.35 mM H₂O₂ or (B) 7 mM FeSO₄, supplemented with 1 mM GSH as indicated, and incubated at 30 °C for 5 days.

3.4. Decreased activities of mitochondrial and cytosolic Fe–S enzymes in the crd1Δ mutant are not due to decreased glutathione levels

The data discussed above suggest that decreased activities of Fe–S enzymes in the CL mutant lead to glutamate and cysteine deficiencies and, as a result, decreased synthesis of glutathione. However, an alternative explanation for decreased glutathione synthesis is that increased levels of oxidants in the CL mutant damage Fe–S enzymes required for synthesis of glutamate and cysteine [45,46]. To distinguish between these possibilities, activities of aconitase (Aco1) and isopropylmalate isomerase (Leu1) were assayed in WT and crd1Δ cells grown in the presence or absence of 1 mM GSH. As the parental strains carry the leu2Δ null mutation, both WT and crd1Δ were transformed with a low/single copy pRS415 plasmid containing the LEU2 gene. As seen in Fig. 4, Aco1 and Leu1 activities were decreased in the crd1Δ mutant by 62.2 ± 8.9% and 49.97 ± 7.07%, respectively, compared to WT, as previously observed [11]. GSH supplementation to crd1Δ cells did not restore enzyme activities to WT levels, suggesting that oxidative damage is most likely not the cause of decreased activities of mitochondrial and cytosolic Fe–S enzymes.

Fig. 4.

Decreased activities of mitochondrial and cytosolic Fe–S enzymes in the crd1Δ mutant are not rescued by supplementation with GSH. (A) For the aconitase assay (Aco1), cells were grown in SD galactose medium lacking glutamate and methionine/cysteine. (B) For the isopropylmalate isomerase assay (Leu1), WT and crd1Δ were transformed with a low/single copy pRS415 plasmid containing the LEU2 gene and grown in SD galactose medium lacking glutamate, methionine/cysteine, and leucine. Fe–S enzymes were assayed as described in materials and methods. Error bars represent mean ± SD (n = 4, two-tailed paired Student’s t-test, *p < .005).

Taken together, these data suggest that CL deficiency leads to decreased activities of Fe–S enzymes required for the synthesis of glutamate and cysteine, resulting in decreased intracellular concentrations of these amino acids and decreased synthesis of glutathione. Supplementation with glutamate and cysteine, or with glutathione, rescued the growth defect of crd1Δ cells at elevated temperature and sensitivity to the oxidants was rescued by supplementation of glutathione.

4. Discussion

In this study, we show for the first time that the mitochondrial lipid CL is essential for the synthesis of glutathione. We previously determined that CL deficiency results in decreased activity of mitochondrial and cytosolic enzymes that require Fe–S cofactors [11]. These include Fe-S-requiring enzymes that play a role in glutamate and cysteine biosynthesis. In the current report, we show that the CL mutant crd1Δ exhibits deficiencies in glutamate and cysteine and decreased levels of glutathione. Mutant phenotypes of decreased growth at elevated temperature and sensitivity to oxidants are rescued by supplementation with glutamate and cysteine or with glutathione.

To maintain glutathione homeostasis, yeast cells take up glutathione or glutathione precursors from the growth medium or synthesize it through the de novo pathway. In synthetic medium, which lacks glutathione, yeast cells depend entirely on de novo synthesis from glutamate, cysteine, and glycine. Therefore, the most likely explanation for decreased growth of the crd1Δ mutant is that glutathione synthesis is decreased due to a deficiency of glutamate and cysteine (Table 2). Overexpression of YAP1, which leads to increased glutathione biosynthetic enzymes, did not restore growth of the CL mutant at elevated temperature (Fig. 2D) or improve resistance of crd1Δ to iron and H₂O₂ [11], suggesting that decreased growth of the mutant is not a result of a deficiency in glutathione biosynthetic enzymes. Previous studies suggested that PE and CL share overlapping functions [2,47], and slightly higher PE levels were observed in crd1Δ (from 17.71% to 19.58% of total phospholipids) [2]. Therefore, it is possible that PE partially compensates for the CL deficiency of crd1Δ in regard to glutathione synthesis.

The mechanism underlying perturbation of Fe–S biogenesis in CL-deficient cells is not understood. The current study suggests that oxidative damage is not the cause of decreased activities of mitochondrial and cytosolic Fe–S enzymes, as supplementing crd1Δ cells with GSH did not restore activities of aconitase (Aco1) or isopropylmalate isomerase (Leu1) (Fig. 4A and B). Previous studies have shown that the presence of CL in mitochondrial membranes is critical for efficient mitochondrial protein import, which is defective in the CL mutant [1,48,49]. Therefore, one possible explanation for decreased Fe–S biogenesis in crd1Δ is that import of one or more proteins involved in the assimilation of mitochondrial Fe–S clusters is decreased. Collectively, these findings suggest that GSH deficiency in the CL mutant is due to decreased Fe–S biogenesis in the mitochondria and cytosol, resulting in depletion of GSH precursors glutamate and cysteine (Fig. 5).

Fig. 5.

Perturbation of GSH metabolism in crd1Δ. In the proposed model, loss of CL leads to decreased mitochondrial and cytosolic Fe–S biogenesis resulting in reduced activities of Fe–S enzymes, including aconitase (Aco1) in the mitochondria and sulfite reductase (SR) in the cytosol. Decreased activity of Aco1 leads to reduced levels of α-ketoglutarate, a key substrate for glutamate biosynthesis. Decreased activity of SR leads to a reduction in intracellular cysteine levels. Decreased levels of both glutamate and cysteine result in reduced biosynthesis of glutathione, a tripeptide of glutamate, cysteine, and glycine, which subsequently causes increased intracellular oxidative stress in crd1Δ.

The finding that CL deficiency deleteriously impacts amino acid and glutathione synthesis has implications for understanding metabolic dysfunction in Barth syndrome, in which CL synthesis is perturbed. Barth syndrome is characterized by wide disparities in clinical presentation, even among patients with identical tafazzin mutations, suggesting that physiological modifiers affect the clinical outcome [50]. Interestingly, abnormalities in the TCA cycle in CL-deficient yeast cells necessitate anaplerotic pathways to replenish TCA cycle metabolites, suggesting that these pathways may be modifiers of the Barth syndrome phenotype [51]. Similarly, amino acid and glutathione metabolism may serve as potential modifiers of the Barth syndrome phenotype and may identify avenues for rescue of metabolic deficiencies.

Abbreviations:

CL

cardiolipin

BTHS

Barth syndrome

Fe-S

iron-sulfur

WT

wild type

SD

synthetic defined

YPD

yeast extract, peptone, and dextrose

YP

yeast extract and peptone

Aco1

aconitase

Leu1

isopropylmalate isomerase

SR

sulfite reductase

FeSO₄

ferrous sulfate

H₂O₂

hydrogen peroxide

ROS

reactive oxygen species

DTT

dithiothreitol

TCA

tricarboxylic acid cycle

GSH

reduced glutathione

GSSG

glutathione disulfide

qPCR

quantitative real-time PCR