Cardiolipin (CL) is a unique phospholipid which is present throughout the eukaryotic kingdom and is localized in mitochondrial membranes. Saccharomyces cerevisiae cells containing a disruption of CRD1, the structural gene encoding CL synthase, have no CL in mitochondrial membranes. To elucidate the physiological role of CL, we compared mitochondrial functions in the crd1⌬ mutant and isogenic wild type. The crd1⌬ mutant loses viability at elevated temperature, and prolonged culture at 37°C leads to loss of the mitochondrial genome. Mutant membranes have increased phosphatidylglycerol (PG) when grown in a nonfermentable carbon source but have almost no detectable PG in medium containing glucose. In glucose-grown cells, maximum respiratory rate, ATPase and cytochrome oxidase activities, and protein import are deficient in the mutant. The ADP/ATP carrier is defective even during growth in a nonfermentable carbon source. The mitochondrial membrane potential is decreased in mutant cells. The decrease is more pronounced in glucose-grown cells, which lack PG, but is also apparent in membranes containing PG (i.e. in nonfermentable carbon sources). We propose that CL is required for maintaining the mitochondrial membrane potential and that reduced membrane potential in the absence of CL leads to defects in protein import and other mitochondrial functions.1 is a structurally unique phospholipid that carries four acyl groups and two negative charges. It is thus highly hydrophobic and acidic. The biosynthesis of CL occurs in three enzymatic steps (1-3). Phosphatidylglycerolphosphate (PGP) synthase catalyzes the formation of PGP from phosphatidyl-CMP (CDP-diacylglycerol; CDP-DG) and glycerol 3-phosphate. PGP is then dephosphorylated to phosphatidylglycerol (PG) by PGP phosphatase. Eukaryotes and bacteria utilize different reactions to convert PG to CL. In prokaryotes, CL synthase catalyzes a phosphatidyl transfer between two PG molecules (4). This is a near-equilibrium (transesterification) reaction that is mainly controlled by substrate availability. In contrast, eukaryotic CL synthase catalyzes a phosphatidyl transfer from CDP-DG to PG (5-7). This is an irreversible reaction that involves cleavage of a high energy anhydride bond. This reaction can take place in the presence of low substrate concentration and is mainly regulated by CL synthase activity. The differences in these reactions probably reflect different functions of PG and CL in prokaryotes and mitochondria.In Escherichia coli, the enzymes that catalyze the synthesis of CL have been characterized biochemically, and the genes encoding these enzymes have been cloned. Although disruption of the cls gene (encoding CL synthase) is not lethal, bacterial strains bearing a null allele of pgsA (encoding PGP synthase) are inviable (8, 9). Interestingly, bacterial cls null mutants do synthesize CL, presumably by another enzyme. These experiments suggest that the anionic phospholipids PG and/or CL are essential for bacterial viability.

SummaryIn 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

Barth syndrome is a genetic disorder that is caused by different mutations in the TAZ gene G4.5. The yeast gene TAZ1 is highly homologous to human TAZ, and the taz1⌬ mutant has phospholipid defects similar to those observed in Barth syndrome cells, including aberrant cardiolipin species and decreased cardiolipin levels. Subcellular fractionation studies revealed that Taz1p is localized exclusively in mitochondria, which supports the theory that tafazzins are involved in cardiolipin remodeling. Because cardiolipin plays an important role in respiratory function, we measured the energy transformation and osmotic properties of isolated mitochondria from the taz1⌬ mutant. Energy coupling in taz1⌬ mitochondria was dependent on the rate of oxidative phosphorylation, as coupling was diminished when NADH was used as a respiratory substrate but was unaffected when ethanol was the substrate. Membrane stability was compromised in taz1⌬ mitochondria exposed to increased temperature and hypotonic conditions. Mitochondria from taz1⌬ also displayed decreased swelling in response to ATP, which induces the yeast mitochondrial unspecific channel, and to alamethicin, a membrane-disrupting agent. Coupling was measured in taz1⌬ cells containing different splice variants of the human TAZ gene. Only the variant that restores wild type cardiolipin synthesis (lacking exon 5) restored coupling in hypotonic conditions and at elevated temperature. These findings may shed light on the mitochondrial deficiencies observed in Barth syndrome.

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SummaryCardiolipin (CL) is a unique dimeric phospholipid localized primarily in the mitochondrial membrane. In eukaryotes, the enzyme CL synthase catalyses the synthesis of CL from two lipid substrates, CDP-diacylglycerol and phosphatidylglycerol. In earlier studies, we reported the purification of CL synthase from Saccharomyces cerevisiae and the cloning of the gene CRD1 (previously called CLS1) that encodes the enzyme. Because CL is an important component of the mitochondrial membrane, knowledge of its regulation will provide insight into the biogenesis of this organelle. To understand how CL synthesis is regulated, we analysed CRD1 expression by Northern blot analysis of RNA extracted from cells under a variety of growth conditions. CRD1 expression is regulated by mitochondrial development factors. CRD1 levels were 7-to 10-fold greater in stationary than in logarithmic growth phase, and threefold greater in wildtype than in 0 mutants. Expression was somewhat elevated during growth in glycerol/ethanol versus glucose media. In contrast, CRD1 expression was not regulated by the phospholipid precursors inositol and choline, and was not altered in the regulatory mutants ino2, ino4 and opi1. Mutations in cytochrome oxidase assembly, which led to reduced Crd1p enzyme activity, did not affect CRD1 expression. The crd1 null mutant makes a truncated CRD1 message. Although the null mutant can grow on both fermentable and non-fermentable carbon sources at lower temperatures, it cannot form colonies at 37ЊC. In conclusion, CRD1 expression is controlled by factors affecting mitochondrial development, but not by the phospholipid precursors inositol and choline. Expression of CRD1 is essential for growth at elevated temperatures, suggesting that either CL or Crd1p is required for an essential cellular function.