Absence of Cardiolipin in the crd1 Null Mutant Results in Decreased Mitochondrial Membrane Potential and Reduced Mitochondrial Function

Abstract

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.

Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria

“…The absence of CL in Dcls1 mitochondria would also be evidenced on whole cells by a cytofluorometric assay on spheroplasts stained with the CL-selective fluorescent probe nonyl acridine orange (NAO; Figure 1d, lower panel). A parallel staining of the cells with the mitochondrial membrane potential (DC m )-sensitive probe DiOC (6) 3 showed that the DC m was reduced, but not completely collapsed, in the Dcls1 strain (Figure 1d, upper panel), predicting that the bioenergetic capacity of Dcls1 mitochondria is moderately altered.…”

“…For DC m measurements, spheroplasts obtained from zymoliase treatment of the cells were incubated for 30 min in the presence of 5 nM DiOC (6) 3 in the absence or in the presence of 10 mM fluorocarbonyl cyanide m-chlorophenylhydrazone (FCCP), and fluorescence was detected at 530730 nm in the FL1 channel. For CLcontent measurements, spheroplasts were incubated for 30 min in the presence of 1 mM NAO and fluorescence was detected at 585740 nm in the FL2 channel.…”

“…It has been known for long that it is associated with mitochondria and, more specifically, with proteins driving oxidative phosphorylation. 1,2 CL has been shown to copurify with different mitochondrial proteins like cytochrome c oxidase, 3,4 the adenine nucleotides carrier, 5,6 the F 0 F 1 -ATP synthase, 7,8 the orthophosphate carrier 9 and the bc 1 complex. 4 For most of them, CL has also been shown to be required for their optimal function.…”

Loss of Function ofKRE5Suppresses Temperature Sensitivity of Mutants Lacking Mitochondrial Anionic Lipids

“…CL plays a key role in mitochondrial bioenergetics (Jiang et al, 2000; Greenberg, 2000, 2002;Schlame et al, 2000;Pfeiffer et al, 2003) and is also involved in mitochondrial biogenesis (Kawasaki et al, 1999;Jiang et al, 2000). Defective remodeling of CL is associated with Barth syndrome, a severe genetic disorder characterized by cardiomyopathy, neutropenia, skeletal myopathy, and respiratory chain defects (Vreken et al, 2000).…”

“…Disruption of PGS1, the structural gene encoding PGP synthase, results in the complete loss of both PG and CL (Janitor et al, 1996;Chang et al, 1998a). The crd1⌬ mutant, which lacks CL synthase, has no detectable CL but accumulates PG (Jiang et al, 1997;Chang et al, 1998b;Tuller et al, 1998;Jiang et al, 2000;Pfeiffer et al, 2003;Zhong et al, 2004). The human taffazin gene (TAZ1), which is associated with Barth syndrome, encodes a transacylase that may be involved in the remodeling of CL (Xu et al, 2003).…”