Summary: In eukaryotic cells, the acyl species of the phospholipid cardiolipin (CL) are more highly unsaturated than those of the other membrane phospholipids. Defective acylation of CL with unsaturated fatty acids and decreased total CL are associated with Barth syndrome, an X‐linked cardio‐ and skeletal myopathy attributed to a defect in the gene G4.5 (also known as tafazzin). We constructed a yeast mutant (taz1) containing a null mutation in the homologue of the human G4.5 gene. The yeast taz1Δ mutant was temperature sensitive for growth in ethanol as sole carbon source, but grew normally on glucose or glycerol plus ethanol. Total CL content was reduced in the taz1Δ mutant, and monolyso‐CL accumulated. The predominant CL acyl species found in wild‐type cells, C18:1 and C16:1, were markedly reduced in the mutant, whereas CL molecules containing saturated fatty acids were present. Interestingly, CL synthesis increased in the mutant, whereas expression of the CL structural genes CRD1 and PGS1 did not, suggesting that de novo biosynthetic enzyme activities are regulated by CL acylation. These results indicate that the taz1Δ mutant is an excellent genetic tool for the study of CL remodelling and may serve as a model system for the study of Barth syndrome.

The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have shown previously that CL and PE have overlapping functions, and the loss of both is synthetically lethal. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, we hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional mutant crd1Δpsd1Δ containing null alleles of CRD1 (CL synthase) and PSD1 (mitochondrial phosphatidylserine decarboxylase), in which the wild type CRD1 gene is expressed on a plasmid under control of the TET_OFF promoter. In the presence of tetracycline, the mutant exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate for the first time in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion.

Saccharomyces cerevisiae mitochondria contain enzymes required for synthesis of the phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE), which are enriched in mitochondrial membranes. Previous studies indicated that PE may compensate for the lack of CL, and vice versa. These data suggest that PE and CL have overlapping functions and that the absence of both lipids may be lethal. To address this hypothesis, we determined whether the crd1Δ mutant, which lacks CL, was viable in genetic backgrounds in which PE synthesis was genetically blocked. Deletion of the mitochondrial PE pathway gene PSD1 was synthetically lethal with the crd1Δ mutant, whereas deletion of the Golgi and endoplasmic reticulum pathway genes PSD2 and DPL1 did not result in synthetic lethality. A 20-fold reduction in phosphatidylcholine did not affect the growth of crd1Δ cells. Supplementation with ethanolamine, which led to increased PE synthesis, or with propanolamine, which led to synthesis of the novel phospholipid phosphatidylpropanolamine, failed to rescue the synthetic lethality of the crd1Δpsd1Δ cells. These results suggest that mitochondrial biosynthesis of PE is essential for the viability of yeast mutants lacking CL.

The role of cardiolipin in mitochondrial function was studied by comparing the energy-transforming and osmotic properties of mitochondria isolated from the Saccharomyces cerevisiae cardiolipin synthase-null mutant crd1Δ, which has no cardiolipin, and the isogenic wild type. The results indicated that the importance of cardiolipin for energetic coupling strongly depends on the rate of oxidative phosphorylation, which was set by using NADH (maximal rate limited by coupling mechanism) or ethanol (moderate rate limited by electron supply) as a respiratory substrate, or by modulating the steady-state rate of NADH supply. The absence of cardiolipin resulted in only a small effect on oxidative phosphorylation proceeding at a moderate rate, but led to significant uncoupling (decreased ADP/O and increased state 4 respiration) at the maximal rate of respiration. This indicates that cardiolipin prevents rate-dependent uncoupling in the energy-transforming apparatus. This role of cardiolipin may derive from its strong interaction with, and modulation of the function of, respiratory complexes, and from its effects on the physical properties of the membrane. The importance of cardiolipin for mitochondrial osmotic properties was determined by comparing oxidative phosphorylation, release of matrix enzyme, shrinking ability and volume dynamics upon hypotonically induced swelling in crd1Δ and wild-type mitochondria. Opening of the yeast mitochondrial unspecific channel (YMUC) in the wild-type and mutant mitochondria was also tested. It was found that the lack of cardiolipin strongly undermines the osmotic stability of the mitochondrial membrane.

Bipolar affective disorder (manic-depressive illness) is a chronic, severe, debilitating illness affecting 1–2% of the population. The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established. We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae. We show that both drugs affect molecular targets in the inositol metabolic pathway. Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis. Theopi1 mutant, which exhibits constitutive expression ofINO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate. Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms. This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate. Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents.