An unusual phosphatidylethanolamine-utilizing cardiolipin synthase is discovered in bacteria George M. Carman1 Department of Food Science and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, NJ 08901 ardiolipin (also known as diphosphatidylglycerol) is C a four-chained anionic mem- brane phospholipid composed of two phosphatidyl moieties joined by a glycerol link (Fig. 1). It was first isolated from beef heart as a serologically active phospholipid (1), hence the name car- diolipin. In animals, plants, and lower eukaryotic organisms (e.g., yeast), car- diolipin is a component of the inner mitochondrial membrane bilayer, where it plays an important role in the function of enzyme complexes involved in energy transduction and ATP synthesis (2, 3). It is found in the membranes of Gram-positive and Gram-negative bacteria, where it also functions in energy production (2, 3). The nature (i.e., chain length and degree of saturation) of the four acyl groups and the hydrophilic head group of this phos- pholipid have an impact on mitochondrial membrane structure and function, and, by extension, the roles this organelle plays in cell physiology (2, 4). Thus, how car- diolipin is synthesized, remodeled, and metabolized are important areas of in- Fig. 1. Pathways for the synthesis of cardiolipin in E. coli and in yeast. In all organisms, cardiolipin is vestigation. The importance of cardiolipin synthesized by pathways emanating from the liponucleotide CDP-diacylglycerol. In prokaryotic cells (e.g., the bacterium E. coli, Left), the final step in cardiolipin synthesis terminates with the joining of two in mitochondrial function is further em- phosphatidylglycerol molecules, but in eukaryotic cells (e.g., yeast, Right), cardiolipin is made from phasized by the fact that defects in ta- phosphatidylglycerol and a second molecule of CDP-diacylglycerol. In the PNAS article by Tan et al. (8), fazzin-mediated remodeling of the fatty cardiolipin in E. coli is also shown to be synthesized from phosphatidylglycerol and phosphatidyletha- acyl composition of cardiolipin is the nolamine by an unusual cardiolipin synthase encoded by the clsC gene (highlighted in gray). The diagram molecular basis for human Barth syn- shows the names of the genes encoding enzymes catalyzing each step in the phospholipid synthesis drome, a disease characterized by heart pathways. The structure of cardiolipin is shown with the synthetic fatty acyl groups that were used to failure, myopathy, neutropenia, and identify the substrates of the clsC-encoded cardiolipin synthase. CL, cardiolipin; CDP-DAG, CDP-diacylglycerol; growth retardation in children (3, 4). PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PGP, phosphatidylglycero- phosphate; PI, phosphatidylinositol; PS, phosphatidylserine. In all organisms, cardiolipin is synthe- sized from phosphatidylglycerol, a two-chained anionic phospholipid derived Two genes, clsA (9, 10) and clsB (11), mechanism by which ymdB expression from the liponucleotide CDP-diacyl- encode phosphatidylglycerol-dependent stimulates ClsC activity is unclear, but glycerol (Fig. 1). How phosphatidyl- cardiolipin synthases (ClsA and ClsB, re- is thus far unique among cardiolipin glycerol is converted to cardiolipin differs spectively) in E. coli. Although cardiolipin synthases. depending on whether cells are prokaryotic is undetectable in exponential phase ΔclsA To identify the phosphatidylethanol- or eukaryotic. In prokaryotic cells, the ΔclsB double mutant cells, the formation amine-dependent nature of this unusual cardiolipin synthase reaction involves the of the lipid is apparent in stationary phase enzyme, Tan et al. (8) carry out a series of transfer of the phosphatidyl moiety of cells indicating a growth phase-dependent elegant biochemical experiments by using phosphatidylglycerol to a second molecule formation of cardiolipin by the newly synthetic phospholipids with unique fatty discovered clsC gene product ClsC. This of phosphatidylglycerol (5), whereas, in acyl moieties and extracts of cells ex- eukaryotic cells, CDP-diacylglycerol do- gene–enzyme relationship was identified pressing plasmid-born clsC-ymdB but nates its phosphatidyl moiety to phospha- by informatics, and confirmation was lacking the ability to synthesize phospha- tidylglycerol to form cardiolipin (6, 7) (Fig. obtained through biochemical experiments Δ 1). In PNAS, Tan et al. (8) use a combina- that included mutagenesis of the enzyme tidylglycerol (e.g., a pgsA mutation) and Δ Δ Δ tion of genetic and biochemical approaches active site (e.g., HKD phospholipase cardiolipin (e.g., clsA clsB clsC to discover an unusual cardiolipin synthase D motifs characteristic of prokaryotic in the bacterium Escherichia coli by which cardiolipin synthases). Maximum expres- phosphatidylethanolamine, a zwitterionic sion of clsC-encoded activity was also Author contributions: G.M.C. wrote the paper. membrane phospholipid, provides the dependent on the product of its neigh- The author declares no conflict of interest. phosphatidyl group to phosphatidylglycerol boring gene, ymdB, but only when both are See companion article on page 16504. to form cardiolipin (Fig. 1). transcribed from the same operon. The 1E-mail: [email protected]. 16402–16403 | PNAS | October 9, 2012 | vol. 109 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1214783109 Downloaded by guest on September 27, 2021 COMMENTARY ΔymdB mutations). By using state-of-the- glycerol, thus expanding the available pool PGS1, CRD1, CHO1, and PIS1, respec- art collision-induced dissociation dual MS of cardiolipin. tively; Fig. 1). Thus, a phosphatidyl- to analyze reaction products, Tan et al. (8) A similar scenario might hold for ethanolamine-dependent reaction would rule out the involvement of CDP-diacyl- eukaryotic cells (e.g., yeast) in which car- allow cardiolipin synthesis under con- glycerol- and phosphatidate-dependent diolipin synthesis is limited by the amounts ditions in which CDP-diacylglycerol is reactions and confirmed that the product limited given that phosphatidylethanol- cardiolipin was composed of fatty acyl amine is an abundant phospholipid. In groups derived from synthetic phosphati- Tan et al. use a fact, a physiological connection between dylglycerol (e.g., 12:0 and 13:0 acyl chains) combination of genetic phosphatidylethanolamine and cardiolipin and synthetic phosphatidylethanolamine has been established in regulating mito- (e.g., 14:1 and 17:0 acyl chains). Thus, and biochemical chondrial function in yeast because loss the power of MS in defining structure of phosphatidylethanolamine synthesis and reaction mechanism was central in approaches to discover (e.g., psd1Δ mutation) is synthetically making this discovery. lethal with the loss of cardiolipin synthesis Although the physiological significant of an unusual cardiolipin (crd1Δ mutation) (12). Whether the basis this phosphatidylethanolamine-dependent for this connection is because phosphati- cardiolipin synthase has yet to be defined, synthase in the dylethanolamine may be used to synthe- there are significant implications that size cardiolipin is not known, but this might be considered. For example, in bacterium Escherichia coli. observation provides impetus for examin- exponential-phase E. coli cells, ample ing a phosphatidylethanolamine-depen- phosphatidylglycerol is most likely avail- dent cardiolipin synthase in yeast and in able via energy dependent formation for of phosphatidylglycerol and CDP-dia- higher eukaryotes. utilization by ClsA and ClsB to make cylglycerol. In particular, CDP-diacyl- Finally, the work of Tan et al. (8) also cardiolipin. However, in stationary phase, glycerol is a minor phospholipid because points to a need to reexamine the mode of in which cardiolipin levels rise while de it is used by four phospholipid synthesis cardiolipin synthesis in the many bacteria novo synthesis of phosphatidylglycerol enzymes (e.g., phosphatidylglycero- with multiple cls homologues identified stops, ClsC can use the ample supply of phosphate synthase, cardiolipin synthase, mainly by informatics and assayed in crude phosphatidylethanolamine as the donor of phosphatidylserine synthase, and phos- extracts assuming involvement of two a phosphatidyl moiety to phosphatidyl- phatidylinositol synthase, encoded by phosphatidylglycerol molecules. 1. Pangborn MC (1942) Isolation and purification of a se- 5. Hirschberg CB, Kennedy EP (1972) Mechanism of the 9. Nishijima S, et al. (1988) Disruption of the Escherichia rologically active phospholipid from beef heart. 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