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Phosphatidylglycerol (PG) is a precursor for the biosynthesis of cardiolipin and a signaling molecule required for various cellular functions. PG is subjected to remodeling subsequent to its de novo biosynthesis in mitochondria to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. Yet, a gene encoding a mitochondrial LPG acyltransferase has not been identified. In this report, we identified a novel function of the human cardiolipin synthase (hCLS1) in regulating PG remodeling. In addition to the reported cardiolipin synthase activity, the recombinant hCLS1 protein expressed in COS-7 cells and Sf-9 insect cells exhibited a strong acyl-CoA-dependent LPG acyltransferase activity, which was further confirmed by purified hCLS1 protein overexpressed in Sf-9 cells. The recombinant hCLS1 displayed an acyl selectivity profile in the order of in the order of C18:1 > C18:2 > C18:0 > C16:0, which is similar to that of hCLS1 toward PGs in cardiolipin synthesis, suggesting that the PG remodeling by hCLS1 is an intrinsic property of the enzyme. In contrast, no significant acyltransferase activity was detected from the recombinant hCLS1 enzyme toward lysocardiolipin which shares a similar structure with LPG. In support of a key function of hCLS1 in PG remodeling, overexpression of hCLS1 in COS-7 cells significantly increased PG biosynthesis concurrent with elevated levels of cardiolipin without any significant effects on the biosynthesis of other phospholipids. These results demonstrate for the first time that hCLS1 catalyzes two consecutive steps in cardiolipin biosynthesis by acylating LPG to PG and then converting PG to cardiolipin.

Apoptosis has been identified recently as a component of many cardiac pathologies. However, the potential triggers of programmed cell death in the heart and the involvement of specific metabolic pathway(s) are less well characterized. Detachment of cytochrome c from the mitochondrial inner membrane is a necessary first step for cytochrome c release into the cytosol and initiation of apoptosis. The saturated long chain fatty acid, palmitate, induces apoptosis in rat neonatal cardiomyocytes and diminishes content of the mitochondrial anionic phospholipid, cardiolipin. These changes are accompanied by 1) acyl chain saturation of phosphatidic acid and phosphatidylglycerol, 2) large increases in the levels of these two phospholipids, and 3) a decline in cardiolipin synthesis. Although cardiolipin synthase activity is unchanged, saturated phosphatidylglycerol is a poor substrate for this enzyme. Under these conditions, decreased cardiolipin synthesis and release of cytochrome c are directly and significantly correlated. The results suggest that phosphatidylglycerol saturation and subsequent decreases in cardiolipin affect the association of cytochrome c with the inner mitochondrial membrane, directly influencing the pathway to cytochrome c release and subsequent apoptosis.

Cardiolipin is a unique dimeric phospholipid, which is present throughout the eukaryotic kingdom and is specifically localized in mitochondrial membranes. It is widely believed that mitochondria possess an essential requirement for this phospholipid. To determine whether cardiolipin is essential for yeast growth, we generated a cardiolipin synthase null mutant by disrupting the CLS1 gene (open reading frame YDL142c on chromosome IV) of Saccharomyces cerevisiae. Biochemical analysis of the mutant indicated that it had no cardiolipin synthase activity and no cardiolipin in its membranes. The enzyme phosphatidylglycerolphosphate synthase, which catalyses the committed step of the cardiolipin pathway, remained unaffected in the null mutant. Haploid cells containing the null allele are viable in media containing glucose, galactose or glycerol/ethanol as the sole carbon source, although growth in galactose or glycerol/ethanol is somewhat reduced in the mutant compared with the wild type. These results indicate that cardiolipin is not essential for the growth of S. cerevisiae in fermentable or non-fermentable carbon sources.

Cardiolipin was first isolated from beef heart and was shown to contain an unusually high content of linoleic acid ester residues. Cardiolipin is found throughout the eukaryotes including animals, plants and fungi. In mammalian tissue and in yeast, cardiolipin is found exclusively in mitochondria. Mitochondrial synthesis of cardiolipin utilizes phosphatidylglycerol and CDP-diacylglycerol as substrates in a reaction which requires a divalent cation (Mg2+, Mn2+ or Co2+). Cardiolipin synthase has been purified to near-homogeneity from rat liver by solubilization with Zwittergent 3-14 followed by FPLC anion exchange, gel permeation and chromatofocusing steps. Cardiolipin synthase has a molecular mass of 50 kDa, a pH optimum of 8.0, and requires added phospholipids (phosphatidylethanolamine and cardiolipin) and 4 mM Co2+ for optimal activity. Except for the effects of divalent cations and the requirement for phospholipids, little is known about the regulation of cardiolipin synthase. Cardiolipin deficiency in aging mitochondria has been linked to decreased metabolite transport across the inner membrane. Both cardiolipin levels and cardiolipin synthase activity are increased in hyperthyroidism and decreased in hypothyroidism suggesting regulation by thyroid hormone. Mammalian cardiolipin synthase has not been sequenced or cloned and its biological role in mitochondria is not yet fully understood.

The effect of administration of exogenous thyroxine on mitochondrial phosphatidylglycerol content and biosynthesis was investigated in rat heart ventricles. Rats were treated for 5 consecutive days with thyroxine (250 mg/kg body weight) and on the sixth day after an overnight fast the mass of ventricular mitochondrial phosphatidylglycerol and cardiolipin content were determined. Saline-treated animals served as controls. Thyroxine treatment did not affect body weight but increased heart weight 30% compared with controls. In addition, the ratio of heart weight/body weight (x 1000) was increased from 0.69 in controls to 0.89 in thyroxine-treated rats consistent with this model. Thyroxine-treatment resulted in a 34% increase (P < 0.05) in phosphatidylglycerol and a 23% increase (P < 0.05) in cardiolipin content in ventricular mitochondrial fractions compared with controls. The mechanism for the increase in ventricular mitochondrial phosphatidylglycerol was investigated. Phosphatidic acid:cytidine-5'-triphosphate-1,2-diacylglycerol cytidylyltransferase and phosphatidylglycerolphosphate phosphatase activities were unaltered in the ventricular mitochondria of thyroxine-treated rats. In contrast, phosphatidylglycerolphosphate synthase activity was increased 3.5-fold (P < 0.05) in these mitochondrial fractions compared with controls. As a control for the effectiveness of thyroxine on mitochondria, cardiolipin synthase activity was determined. A 2.8-fold increase (P < 0.05) in cardiolipin synthase activity was observed in ventricular mitochondrial fractions of thyroxine-treated rats compared with controls. We postulate that thyroxine-treatment of rats produces an increase in the pool size of ventricular mitochondrial phosphatidylglycerol and that the mechanism is an increase in phosphatidylglycerolphosphate synthase activity.

Escherichia coli cardiolipin synthase catalyzes the conversion of two phosphatidylglycerol molecules to cardiolipin and glycerol. This enzyme was amplified in strain BL21(DE3) bearing recombinant plasmid pLR3, which was itself constructed by inserting the cls gene downstream from a T7 RNA promoter. Membranes from BL21(DE3)/pLR3 have over 1200 times more cardiolipin synthase activity than do comparable membranes from wild type cells. The enzyme was purified to homogeneity by extraction with Triton X-114 and chromatography on DEAE-cellulose. The purified enzyme migrated as a single band (46 kDa) on SDS-PAGE. This, along with SDS-PAGE analysis of induced protein, supports the notion that cls is the structural gene for cardiolipin synthase. Cardiolipin synthase activity was determined in a mixed micelle assay in which phosphatidyl[2-3H]glycerol was the substrate. The enzyme is inhibited by the product of the reaction, cardiolipin, and by phosphatidate. However, it is not inhibited by two other anionic phosphoglycerides, phosphatidylinositol and bis-phosphatidate. Phosphatidylethanolamine partially offsets inhibition by cardiolipin but not by phosphatidate. Magnesium chloride has the opposite effect. Cardiolipin inhibition of cardiolipin synthase probably plays an important role in regulating cardiolipin synthesis in E. coli.

In a previous work temporal succession of two sheathed, filamentous methanotrophic γ-Proteobacteria, Crenothrix polyspora and Clonothrix fusca, was observed by analyzing the microbial community of an artesian well. Although a correlation between physical-chemical environmental parameters and growth of these microorganisms was suggested, the link with the microbial physiology remained elusive. In this study we demonstrate that phosphatidylethanolamine and cardiolipin, generally abundant in most γ-Proteobacteria, could not be detected in C. fusca membranes by thin-layer chromatography. The absence of cardiolipin was supported by results of enzymatic assay of cardiolipin synthase activity and by Southern blot analysis that failed to detect cls homologous sequences in the genomic DNA of this microorganism. The absence of phosphatidylethanolamine and cardiolipin might account for requirement of millimolar concentrations of magnesium and calcium for growth, and for massive over-growth of this microorganism during the winter season when the water levels of magnesium and calcium increased in the artesian well. © 2007 Taylor & Francis.

Cardiolipin synthase catalyzes the synthesis of the mitochondrial phospholipid cardiolipin. Cardiolipin synthase is a unique membrane-bound enzyme in that it utilizes two phospholipids, both insoluble in water, as substrates. Kinetic analysis suggests that the enzyme forms a ternary complex with the two lipid substrates, and that a divalent metal ion directly associates with cardiolipin synthase to form the active enzyme. While little is known about the regulation of cardiolipin synthase in yeast, activity is reduced in mutants in which the mitochondrial genome is deleted, and in mutants with defective respiratory complexes. In p0 mutants, which contain no mitochondrial DNA and are defective in the assembly of many mitochondrial membrane protein complexes, cardiolipin synthase activity is reduced by 50%. Mutants defective in respiratory complexes, particularly those incapable of cytochrome oxidase assembly, also have reduced cardiolipin synthase activity. Thus it is likely that respiration and cardiolipin formation are interdependent. The enzyme was recently purified from the budding yeast Saccharomyces cerevisiae. Enzyme activity was associated with a 25-30-kDa protein. The amino acid sequence of this protein, combined with the availability of the complete yeast genome sequence, will hopefully lead to the identification of the structural gene for this enzyme in the near future.

Publisher Summary In Escherichia coli (E.coli), cardiolipin is mainly synthesized by cardiolipin synthase, encoded by the cls gene, via condensation of two molecules of phosphatidylglycerol, also yielding one molecule of glycerol. This contrasts with the eukaryotic enzymes that condense CDPdiacylglycerol and phosphatidylglycerol to form cardiolipin and cytidine monophosphate (CMP). The biological implication of the metabolically uneconomical pathway in E. coli is unknown. The E. coli cardiolipin synthase is present exclusively in the envelope (crude membrane) fraction of the cell. The E. coli cls null mutants that are completely devoid of cardiolipin synthase activity grow well and form small amounts of cardiolipin, suggesting that E. coli possesses an alternative minor pathway to form cardiolipin. The most likely candidate responsible for this second pathway is phosphatidylserine synthase. It has been suggested that cardiolipin synthase may catalyze practically reversible reactions, and its relaxed substrate specificity may allow the formation of lyso- and dilysocardiolipins from 2-acyllysophosphatidylglycerol and a series of analogs of phosphatidylglycerol and cardiolipin if high concentrations of the appropriate straight-chain sugar alcohols are present in the culture medium.

Cardiolipin is a major mitochondrial membrane lipid and plays a vital role in mitochondrial function. The effect of thyroxine on the activity of liver mitochondrial cardiolipin synthase was examined in this study. Treatment with thyroxine (250 micrograms/100 g) for 5 days increased cardiolipin synthase activity by 52%. Mitochondrial levels of cardiolipin appear to be regulated in part by the effect of thyroid hormone on the activity of cardiolipin synthase.

Background Monolysocardiolipin acyltransferase (MLCL AT) catalyzes the acylation of monolysocardiolipin to cardiolipin in mammalian tissues. We previously reported that cardiac cardiolipin levels, MLCL AT and cardiolipin synthase activities were all elevated in rats made hyperthyroid by thyroxine treatment. In this study, we examined if cardiac mitochondrial MLCL AT activity was dependent upon the biosynthesis and level of cardiolipin in the heart. Rat heart mitochondrial MLCL AT activity was determined under conditions in which the levels of cardiac cardiolipin and cardiolipin synthase activity were either reduced or unaltered using four different disease models in the rat. In addition, these parameters were examined in a murine model of cardiac cell differentiation.

When cultured in the presence of 600 mM D-mannitol, Escherichia coli K-12 cells synthesized two novel phospholipids. The identities of these compounds are postulated to be phosphatidylmannitol and diphosphatidylmannitol, the sugar alcohol analogs of phosphatidylglycerol and cardiolipin, respectively. The nonacylated glycerol moieties of the normal acidic phospholipids were substituted by D-mannitol. The formation of the analogs was significantly enhanced when strains harboring the pss-1 allele, a temperature-sensitive mutation in phosphatidylserine synthase (Ohta and Shibuya, J. Bacteriol. 132:434-443, 1977), were grown at 42 degrees C, and the accumulation of the analogs was maximum in late stationary phase; more than 90% of the total cellular lipids were these novel phospholipids. Strains with a defective cardiolipin synthase (Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) failed to form the analog lipids, whereas cells with increased cardiolipin synthase activity due to the presence of a pBR322-derived recombinant plasmid containing the structural gene for cardiolipin synthase produced more mannitol lipids than wild-type strains. These observations and the structures of the analog lipids indicated that cardiolipin synthase participates in the formation of these novel phospholipids. We suggest that reversible alcoholysis and condensation, in addition to low substrate specificity of the enzyme, are the mechanisms involved in this process. Addition to the medium of other straight-chain alditols, D-arabitol, ribitol, xylitol, erythritol, and L-threitol also yielded pairs of novel phospholipids, whereas sorbitol or galactitol produced only one analog in small quantities. These acidic phospholipid analogs have not been reported in any living system. They should be useful in the study of structure-function relationships of phospholipids and in manipulating the structures of various membrane systems.

The cls gene responsible for cardiolipin synthesis in Escherichia coli K-12 was cloned in a 5-kilobase-pair DNA fragment inserted in a mini-F vector, pML31, and then subcloned into a 2.0-kilobase-pair fragment inserted in pBR322. The initial selection of the gene was accomplished in a cls pss-1 double mutant that had lesions in both cardiolipin and phosphatidylserine synthases and required either the cls or the pss gene product for normal growth at 42 degrees C in a broth medium, NBY, supplemented with 200 mM sucrose. The cloned gene was identified as the cls gene by the recovery and amplification of both cardiolipin and cardiolipin synthase in a cls mutant as well as by the integration of a pBR322 derivative into its genetic locus at 27 min on the chromosome of a polA1 mutant. The maxicell analysis indicated that a protein of molecular weight 46,000 is the gene product. The cls gene is thus most likely the structural gene coding for cardiolipin synthase. Hybrid plasmids of high copy numbers containing the cls gene were growth inhibitory to pss-I mutants under the above selective conditions, whereas they inhibited neither the growth of pss-I mutants at 30 degrees C nor that of pss+ strains at any temperature. Amplification of cardiolipin synthase activity was observed, but was not proportional to the probable gene dosage (the enzyme activity was at most 10 times that in wild-type cells), and cardiolipin synthesis in vivo was at the maximum 1.5 times that in wild-type strains, implying the presence in E. coli cells of a mechanism that avoids cardiolipin overproduction, which is possibly disadvantageous to proper membrane functions.